Bridging the Gap Between Our Understanding of AML Pathogenesis and the Development of Targeted

Therapies

Montreh Tavakkoli

Submitted in partial fulfillment of therequirements for the degree of

Masters of Artsin the Graduate School of Arts and Sciences

Program in BiotechnologyDepartment of Biological Sciences

COLUMBIA UNIVERSITY

2014

copy 2014Montreh Tavakkoli

All Rights Reserved

ABSTRACT

Bridging the Gap Between Our Understanding of AML Pathogenesis and the Development of Targeted Therapies

Montreh Tavakkoli

Acute myeloid leukemia (AML) is the most common acute leukemia diagnosed in the US with an annual incidence of ~15000 per year The median age of diagnosis is 67 years however AML afflicts individuals of all ages Within the past 4 decades only modest improvements have been made in the treatment of AML However the advent of DNA sequencing technologies fluorescence-activated cell sorting flow cytometry and immunodeficient murine models have significantly improved our understanding of the molecular and cellular changes that promote the development of AML The purpose of my thesis is to explain our current understanding of malignant transformation in AML and to describe how this knowledge has aided the clinical assessment and treatment of this disease In order to demonstrate this I will provide an introduction to the epidemiology and clinical manifestations of AML the molecular mechanisms underlying its pathogenesis and new methods to risk-stratify and treat the disease I will then provide a thorough discussion on normal hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the cancer stem cell theory) and will conclude with a brief summary on a novel leukemic stem cell-directed therapy that we are currently developing in our laboratory For the first time in over 40 years drastic changes are underway in the way we evaluate and treat AML

TABLE OF CONTENTS

List of Figures ii

List of Tables iii

List of Abbreviations iv-v

Acknowledgments vi

Dedications vii

1 Introduction 1

2 Acute Myeloid Leukemia

21 Epidemiology 5

22 Clinical Manifestations 6

23 Pathogenesis 7

24 Implications of Molecular Aberrations on Risk Stratification 11

25 Targeting Recurrent MolecularChromosomal Aberrations 15

3 Normal Hematopoiesis

31 Hierarchical Organization of the Hematopoietic System 23

32 HSC Immunophenotyping 25

4 AML and the Cancer Stem Cell Theory

41 Proof of LSCs 29

42 LSC Cell of Origin 32

43 Current Model for the Hierarchical Organization of AML 34

5 AML ndash Therapeutic Implications of the Cancer Stem Cell Theory

51 LSC Resistance to Conventional Therapies 38

52 Therapeutic Targeting of CD99 39

6 Conclusions 43

7 Figures 45

8 Tables 49

9 References 51

List of Figures

Figure 1 Incidence of AML in the USFigure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesisFigure 3 Normal hematopoiesisFigure 4 Malignant hematopoiesis in AML Figure 5 Proposed model for relapse following conventional chemotherapyFigure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs and preleukemic progenitors and thus produces a novel heterogeneous group of LSCs in the bone marrow

ii

List of Tables

Table 1 FAB classification of AML subtypes (Arber et al 2003)Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)Table 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

iii

List of Abbreviations

AML acute myeloid leukemiaCFU-S colony-forming unit spleenCSC cancer stem cellAYAs adolescents and young adultsMDS myelodysplastic syndromeOS overall survivalBM bone marrowFAB French American BritishWBC white blood cellRBC red blood cellFLT3-ITD fms-like tyrosine kinase 3-internal tandem duplicationCBF core binding factorMYH myosin-heavy chainPML-RARα promyelocytic leukemia-retinoic acid receptor alphaFPD familial platelet disorderRUNX1 runt-related transcription factor 1BCL-2 B-cell lymphoma 2IDH12 isocitrate dehydrogenase 12MPD myeloproliferative disorderTET2 tet methylcytosine dioxygenase 2MLL mixed-lineage leukemiaASXL1 additional sex combs like 1DNMT3A DNA methyltransferase 3AIL-3 interleukin-3CN cytogenetically normalNCCN National Comprehensive Cancer NetworkNPM1 nucleophosmin protein member 1CEBPα CCAATenhancer-binding protein alphaECOG Eastern Cooperative Oncology GroupPTD partial tandem duplicationPHF6 PHD finger protein 6MK monosomal karyotypeWT1 wilmrsquos tumor 1CR complete remissionDFS disease-free survivalHSCT hematopoietic stem cell transplantationHDAC high-dose cytarabineHLA human leukocyte antigenHSC hematopoietic stem cellHPA hypomethylating agentPI3K phosphatidylinositol-45-bisphosphate 3-kinaseAKT protein kinase BMAPK mitogen-activated protein kinaseSTAT5 signal transducer and activator of transcription 5

iv

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

copy 2014Montreh Tavakkoli

All Rights Reserved

ABSTRACT

Bridging the Gap Between Our Understanding of AML Pathogenesis and the Development of Targeted Therapies

Montreh Tavakkoli

Acute myeloid leukemia (AML) is the most common acute leukemia diagnosed in the US with an annual incidence of ~15000 per year The median age of diagnosis is 67 years however AML afflicts individuals of all ages Within the past 4 decades only modest improvements have been made in the treatment of AML However the advent of DNA sequencing technologies fluorescence-activated cell sorting flow cytometry and immunodeficient murine models have significantly improved our understanding of the molecular and cellular changes that promote the development of AML The purpose of my thesis is to explain our current understanding of malignant transformation in AML and to describe how this knowledge has aided the clinical assessment and treatment of this disease In order to demonstrate this I will provide an introduction to the epidemiology and clinical manifestations of AML the molecular mechanisms underlying its pathogenesis and new methods to risk-stratify and treat the disease I will then provide a thorough discussion on normal hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the cancer stem cell theory) and will conclude with a brief summary on a novel leukemic stem cell-directed therapy that we are currently developing in our laboratory For the first time in over 40 years drastic changes are underway in the way we evaluate and treat AML

TABLE OF CONTENTS

List of Figures ii

List of Tables iii

List of Abbreviations iv-v

Acknowledgments vi

Dedications vii

1 Introduction 1

2 Acute Myeloid Leukemia

21 Epidemiology 5

22 Clinical Manifestations 6

23 Pathogenesis 7

24 Implications of Molecular Aberrations on Risk Stratification 11

25 Targeting Recurrent MolecularChromosomal Aberrations 15

3 Normal Hematopoiesis

31 Hierarchical Organization of the Hematopoietic System 23

32 HSC Immunophenotyping 25

4 AML and the Cancer Stem Cell Theory

41 Proof of LSCs 29

42 LSC Cell of Origin 32

43 Current Model for the Hierarchical Organization of AML 34

5 AML ndash Therapeutic Implications of the Cancer Stem Cell Theory

51 LSC Resistance to Conventional Therapies 38

52 Therapeutic Targeting of CD99 39

6 Conclusions 43

7 Figures 45

8 Tables 49

9 References 51

List of Figures

Figure 1 Incidence of AML in the USFigure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesisFigure 3 Normal hematopoiesisFigure 4 Malignant hematopoiesis in AML Figure 5 Proposed model for relapse following conventional chemotherapyFigure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs and preleukemic progenitors and thus produces a novel heterogeneous group of LSCs in the bone marrow

ii

List of Tables

Table 1 FAB classification of AML subtypes (Arber et al 2003)Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)Table 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

iii

List of Abbreviations

AML acute myeloid leukemiaCFU-S colony-forming unit spleenCSC cancer stem cellAYAs adolescents and young adultsMDS myelodysplastic syndromeOS overall survivalBM bone marrowFAB French American BritishWBC white blood cellRBC red blood cellFLT3-ITD fms-like tyrosine kinase 3-internal tandem duplicationCBF core binding factorMYH myosin-heavy chainPML-RARα promyelocytic leukemia-retinoic acid receptor alphaFPD familial platelet disorderRUNX1 runt-related transcription factor 1BCL-2 B-cell lymphoma 2IDH12 isocitrate dehydrogenase 12MPD myeloproliferative disorderTET2 tet methylcytosine dioxygenase 2MLL mixed-lineage leukemiaASXL1 additional sex combs like 1DNMT3A DNA methyltransferase 3AIL-3 interleukin-3CN cytogenetically normalNCCN National Comprehensive Cancer NetworkNPM1 nucleophosmin protein member 1CEBPα CCAATenhancer-binding protein alphaECOG Eastern Cooperative Oncology GroupPTD partial tandem duplicationPHF6 PHD finger protein 6MK monosomal karyotypeWT1 wilmrsquos tumor 1CR complete remissionDFS disease-free survivalHSCT hematopoietic stem cell transplantationHDAC high-dose cytarabineHLA human leukocyte antigenHSC hematopoietic stem cellHPA hypomethylating agentPI3K phosphatidylinositol-45-bisphosphate 3-kinaseAKT protein kinase BMAPK mitogen-activated protein kinaseSTAT5 signal transducer and activator of transcription 5

iv

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

ABSTRACT

Bridging the Gap Between Our Understanding of AML Pathogenesis and the Development of Targeted Therapies

Montreh Tavakkoli

Acute myeloid leukemia (AML) is the most common acute leukemia diagnosed in the US with an annual incidence of ~15000 per year The median age of diagnosis is 67 years however AML afflicts individuals of all ages Within the past 4 decades only modest improvements have been made in the treatment of AML However the advent of DNA sequencing technologies fluorescence-activated cell sorting flow cytometry and immunodeficient murine models have significantly improved our understanding of the molecular and cellular changes that promote the development of AML The purpose of my thesis is to explain our current understanding of malignant transformation in AML and to describe how this knowledge has aided the clinical assessment and treatment of this disease In order to demonstrate this I will provide an introduction to the epidemiology and clinical manifestations of AML the molecular mechanisms underlying its pathogenesis and new methods to risk-stratify and treat the disease I will then provide a thorough discussion on normal hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the cancer stem cell theory) and will conclude with a brief summary on a novel leukemic stem cell-directed therapy that we are currently developing in our laboratory For the first time in over 40 years drastic changes are underway in the way we evaluate and treat AML

TABLE OF CONTENTS

List of Figures ii

List of Tables iii

List of Abbreviations iv-v

Acknowledgments vi

Dedications vii

1 Introduction 1

2 Acute Myeloid Leukemia

21 Epidemiology 5

22 Clinical Manifestations 6

23 Pathogenesis 7

24 Implications of Molecular Aberrations on Risk Stratification 11

25 Targeting Recurrent MolecularChromosomal Aberrations 15

3 Normal Hematopoiesis

31 Hierarchical Organization of the Hematopoietic System 23

32 HSC Immunophenotyping 25

4 AML and the Cancer Stem Cell Theory

41 Proof of LSCs 29

42 LSC Cell of Origin 32

43 Current Model for the Hierarchical Organization of AML 34

5 AML ndash Therapeutic Implications of the Cancer Stem Cell Theory

51 LSC Resistance to Conventional Therapies 38

52 Therapeutic Targeting of CD99 39

6 Conclusions 43

7 Figures 45

8 Tables 49

9 References 51

List of Figures

Figure 1 Incidence of AML in the USFigure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesisFigure 3 Normal hematopoiesisFigure 4 Malignant hematopoiesis in AML Figure 5 Proposed model for relapse following conventional chemotherapyFigure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs and preleukemic progenitors and thus produces a novel heterogeneous group of LSCs in the bone marrow

ii

List of Tables

Table 1 FAB classification of AML subtypes (Arber et al 2003)Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)Table 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

iii

List of Abbreviations

AML acute myeloid leukemiaCFU-S colony-forming unit spleenCSC cancer stem cellAYAs adolescents and young adultsMDS myelodysplastic syndromeOS overall survivalBM bone marrowFAB French American BritishWBC white blood cellRBC red blood cellFLT3-ITD fms-like tyrosine kinase 3-internal tandem duplicationCBF core binding factorMYH myosin-heavy chainPML-RARα promyelocytic leukemia-retinoic acid receptor alphaFPD familial platelet disorderRUNX1 runt-related transcription factor 1BCL-2 B-cell lymphoma 2IDH12 isocitrate dehydrogenase 12MPD myeloproliferative disorderTET2 tet methylcytosine dioxygenase 2MLL mixed-lineage leukemiaASXL1 additional sex combs like 1DNMT3A DNA methyltransferase 3AIL-3 interleukin-3CN cytogenetically normalNCCN National Comprehensive Cancer NetworkNPM1 nucleophosmin protein member 1CEBPα CCAATenhancer-binding protein alphaECOG Eastern Cooperative Oncology GroupPTD partial tandem duplicationPHF6 PHD finger protein 6MK monosomal karyotypeWT1 wilmrsquos tumor 1CR complete remissionDFS disease-free survivalHSCT hematopoietic stem cell transplantationHDAC high-dose cytarabineHLA human leukocyte antigenHSC hematopoietic stem cellHPA hypomethylating agentPI3K phosphatidylinositol-45-bisphosphate 3-kinaseAKT protein kinase BMAPK mitogen-activated protein kinaseSTAT5 signal transducer and activator of transcription 5

iv

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

TABLE OF CONTENTS

List of Figures ii

List of Tables iii

List of Abbreviations iv-v

Acknowledgments vi

Dedications vii

1 Introduction 1

2 Acute Myeloid Leukemia

21 Epidemiology 5

22 Clinical Manifestations 6

23 Pathogenesis 7

24 Implications of Molecular Aberrations on Risk Stratification 11

25 Targeting Recurrent MolecularChromosomal Aberrations 15

3 Normal Hematopoiesis

31 Hierarchical Organization of the Hematopoietic System 23

32 HSC Immunophenotyping 25

4 AML and the Cancer Stem Cell Theory

41 Proof of LSCs 29

42 LSC Cell of Origin 32

43 Current Model for the Hierarchical Organization of AML 34

5 AML ndash Therapeutic Implications of the Cancer Stem Cell Theory

51 LSC Resistance to Conventional Therapies 38

52 Therapeutic Targeting of CD99 39

6 Conclusions 43

7 Figures 45

8 Tables 49

9 References 51

List of Figures

Figure 1 Incidence of AML in the USFigure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesisFigure 3 Normal hematopoiesisFigure 4 Malignant hematopoiesis in AML Figure 5 Proposed model for relapse following conventional chemotherapyFigure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs and preleukemic progenitors and thus produces a novel heterogeneous group of LSCs in the bone marrow

ii

List of Tables

Table 1 FAB classification of AML subtypes (Arber et al 2003)Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)Table 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

iii

List of Abbreviations

AML acute myeloid leukemiaCFU-S colony-forming unit spleenCSC cancer stem cellAYAs adolescents and young adultsMDS myelodysplastic syndromeOS overall survivalBM bone marrowFAB French American BritishWBC white blood cellRBC red blood cellFLT3-ITD fms-like tyrosine kinase 3-internal tandem duplicationCBF core binding factorMYH myosin-heavy chainPML-RARα promyelocytic leukemia-retinoic acid receptor alphaFPD familial platelet disorderRUNX1 runt-related transcription factor 1BCL-2 B-cell lymphoma 2IDH12 isocitrate dehydrogenase 12MPD myeloproliferative disorderTET2 tet methylcytosine dioxygenase 2MLL mixed-lineage leukemiaASXL1 additional sex combs like 1DNMT3A DNA methyltransferase 3AIL-3 interleukin-3CN cytogenetically normalNCCN National Comprehensive Cancer NetworkNPM1 nucleophosmin protein member 1CEBPα CCAATenhancer-binding protein alphaECOG Eastern Cooperative Oncology GroupPTD partial tandem duplicationPHF6 PHD finger protein 6MK monosomal karyotypeWT1 wilmrsquos tumor 1CR complete remissionDFS disease-free survivalHSCT hematopoietic stem cell transplantationHDAC high-dose cytarabineHLA human leukocyte antigenHSC hematopoietic stem cellHPA hypomethylating agentPI3K phosphatidylinositol-45-bisphosphate 3-kinaseAKT protein kinase BMAPK mitogen-activated protein kinaseSTAT5 signal transducer and activator of transcription 5

iv

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

6 Conclusions 43

7 Figures 45

8 Tables 49

9 References 51

List of Figures

Figure 1 Incidence of AML in the USFigure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesisFigure 3 Normal hematopoiesisFigure 4 Malignant hematopoiesis in AML Figure 5 Proposed model for relapse following conventional chemotherapyFigure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs and preleukemic progenitors and thus produces a novel heterogeneous group of LSCs in the bone marrow

ii

List of Tables

Table 1 FAB classification of AML subtypes (Arber et al 2003)Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)Table 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

iii

List of Abbreviations

AML acute myeloid leukemiaCFU-S colony-forming unit spleenCSC cancer stem cellAYAs adolescents and young adultsMDS myelodysplastic syndromeOS overall survivalBM bone marrowFAB French American BritishWBC white blood cellRBC red blood cellFLT3-ITD fms-like tyrosine kinase 3-internal tandem duplicationCBF core binding factorMYH myosin-heavy chainPML-RARα promyelocytic leukemia-retinoic acid receptor alphaFPD familial platelet disorderRUNX1 runt-related transcription factor 1BCL-2 B-cell lymphoma 2IDH12 isocitrate dehydrogenase 12MPD myeloproliferative disorderTET2 tet methylcytosine dioxygenase 2MLL mixed-lineage leukemiaASXL1 additional sex combs like 1DNMT3A DNA methyltransferase 3AIL-3 interleukin-3CN cytogenetically normalNCCN National Comprehensive Cancer NetworkNPM1 nucleophosmin protein member 1CEBPα CCAATenhancer-binding protein alphaECOG Eastern Cooperative Oncology GroupPTD partial tandem duplicationPHF6 PHD finger protein 6MK monosomal karyotypeWT1 wilmrsquos tumor 1CR complete remissionDFS disease-free survivalHSCT hematopoietic stem cell transplantationHDAC high-dose cytarabineHLA human leukocyte antigenHSC hematopoietic stem cellHPA hypomethylating agentPI3K phosphatidylinositol-45-bisphosphate 3-kinaseAKT protein kinase BMAPK mitogen-activated protein kinaseSTAT5 signal transducer and activator of transcription 5

iv

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

List of Figures

Figure 1 Incidence of AML in the USFigure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesisFigure 3 Normal hematopoiesisFigure 4 Malignant hematopoiesis in AML Figure 5 Proposed model for relapse following conventional chemotherapyFigure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs and preleukemic progenitors and thus produces a novel heterogeneous group of LSCs in the bone marrow

ii

List of Tables

Table 1 FAB classification of AML subtypes (Arber et al 2003)Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)Table 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

iii

List of Abbreviations

AML acute myeloid leukemiaCFU-S colony-forming unit spleenCSC cancer stem cellAYAs adolescents and young adultsMDS myelodysplastic syndromeOS overall survivalBM bone marrowFAB French American BritishWBC white blood cellRBC red blood cellFLT3-ITD fms-like tyrosine kinase 3-internal tandem duplicationCBF core binding factorMYH myosin-heavy chainPML-RARα promyelocytic leukemia-retinoic acid receptor alphaFPD familial platelet disorderRUNX1 runt-related transcription factor 1BCL-2 B-cell lymphoma 2IDH12 isocitrate dehydrogenase 12MPD myeloproliferative disorderTET2 tet methylcytosine dioxygenase 2MLL mixed-lineage leukemiaASXL1 additional sex combs like 1DNMT3A DNA methyltransferase 3AIL-3 interleukin-3CN cytogenetically normalNCCN National Comprehensive Cancer NetworkNPM1 nucleophosmin protein member 1CEBPα CCAATenhancer-binding protein alphaECOG Eastern Cooperative Oncology GroupPTD partial tandem duplicationPHF6 PHD finger protein 6MK monosomal karyotypeWT1 wilmrsquos tumor 1CR complete remissionDFS disease-free survivalHSCT hematopoietic stem cell transplantationHDAC high-dose cytarabineHLA human leukocyte antigenHSC hematopoietic stem cellHPA hypomethylating agentPI3K phosphatidylinositol-45-bisphosphate 3-kinaseAKT protein kinase BMAPK mitogen-activated protein kinaseSTAT5 signal transducer and activator of transcription 5

iv

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

List of Tables

Table 1 FAB classification of AML subtypes (Arber et al 2003)Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)Table 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

iii

List of Abbreviations

AML acute myeloid leukemiaCFU-S colony-forming unit spleenCSC cancer stem cellAYAs adolescents and young adultsMDS myelodysplastic syndromeOS overall survivalBM bone marrowFAB French American BritishWBC white blood cellRBC red blood cellFLT3-ITD fms-like tyrosine kinase 3-internal tandem duplicationCBF core binding factorMYH myosin-heavy chainPML-RARα promyelocytic leukemia-retinoic acid receptor alphaFPD familial platelet disorderRUNX1 runt-related transcription factor 1BCL-2 B-cell lymphoma 2IDH12 isocitrate dehydrogenase 12MPD myeloproliferative disorderTET2 tet methylcytosine dioxygenase 2MLL mixed-lineage leukemiaASXL1 additional sex combs like 1DNMT3A DNA methyltransferase 3AIL-3 interleukin-3CN cytogenetically normalNCCN National Comprehensive Cancer NetworkNPM1 nucleophosmin protein member 1CEBPα CCAATenhancer-binding protein alphaECOG Eastern Cooperative Oncology GroupPTD partial tandem duplicationPHF6 PHD finger protein 6MK monosomal karyotypeWT1 wilmrsquos tumor 1CR complete remissionDFS disease-free survivalHSCT hematopoietic stem cell transplantationHDAC high-dose cytarabineHLA human leukocyte antigenHSC hematopoietic stem cellHPA hypomethylating agentPI3K phosphatidylinositol-45-bisphosphate 3-kinaseAKT protein kinase BMAPK mitogen-activated protein kinaseSTAT5 signal transducer and activator of transcription 5

iv

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

List of Abbreviations

AML acute myeloid leukemiaCFU-S colony-forming unit spleenCSC cancer stem cellAYAs adolescents and young adultsMDS myelodysplastic syndromeOS overall survivalBM bone marrowFAB French American BritishWBC white blood cellRBC red blood cellFLT3-ITD fms-like tyrosine kinase 3-internal tandem duplicationCBF core binding factorMYH myosin-heavy chainPML-RARα promyelocytic leukemia-retinoic acid receptor alphaFPD familial platelet disorderRUNX1 runt-related transcription factor 1BCL-2 B-cell lymphoma 2IDH12 isocitrate dehydrogenase 12MPD myeloproliferative disorderTET2 tet methylcytosine dioxygenase 2MLL mixed-lineage leukemiaASXL1 additional sex combs like 1DNMT3A DNA methyltransferase 3AIL-3 interleukin-3CN cytogenetically normalNCCN National Comprehensive Cancer NetworkNPM1 nucleophosmin protein member 1CEBPα CCAATenhancer-binding protein alphaECOG Eastern Cooperative Oncology GroupPTD partial tandem duplicationPHF6 PHD finger protein 6MK monosomal karyotypeWT1 wilmrsquos tumor 1CR complete remissionDFS disease-free survivalHSCT hematopoietic stem cell transplantationHDAC high-dose cytarabineHLA human leukocyte antigenHSC hematopoietic stem cellHPA hypomethylating agentPI3K phosphatidylinositol-45-bisphosphate 3-kinaseAKT protein kinase BMAPK mitogen-activated protein kinaseSTAT5 signal transducer and activator of transcription 5

iv

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

PBMNC peripheral blood mononuclear cell2-HG 2-hydroxyglutarateDOT1L DOT1-likeMRD minimal residual diseaseCAR chimeric-antigen receptorCD cluster of differentiationMPP multipotent progenitorCLP common lymphoid progenitorNK natural killerCMP common myeloid progenitorGMP granulocyte monocyte progenitorMEP megakaryocyte-erythroid progenitorMLP multilymphoid progenitorLMPP lymphoid-primed multipotential progenitorFACS fluorescence-activated cell sortingsorterLTC-IC long-term culture-initiating cellLDA limiting dilution analysisNOD non-obese diabeticSCID severe combined immunodeficiencySL-IC SCID leukemia-initiating cellNSG NOD SCID gammaIVIG intravenous immunoglobulinENL eleven nineteen leukemiaTIM3 T cell immunoglobulin mucin-3Ab antibodymAb monoclonal antibodyADCC antigen-dependent cellular cytotoxicityB-ALL B-cell acute lymphocytic leukemiaBCR-ABL breakpoint cluster region protein-abelson murine leukemia viral oncogeneHUVEC human umbilical vein endothelial cellUCB umbilical cord blood

v

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Acknowledgements

I am very grateful to my thesis advisors Christopher Y Park MD PhD Principal Investigator in the Human Oncology and Pathogenesis Program and Hematopathologist at the Memorial Sloan-Kettering Cancer Center and Ron Guido Lecturer at Columbia University and Vice President of Regulatory Affairs at Retrophin for devoting their valuable time to my thesis

vi

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

To my parents sister family and friends who have supported me throughout my career and my life

Two roads diverged in a wood and I - I took the one less traveled by And that has made all the difference ndashRobert Frost

vii

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

1 Introduction

Cancer etiologies have been investigated for centuries with the original cause of cancer

being described by Greek philosopher Hippocrates According to his theory of

humorism the body consists of four bodily fluids or humors including yellow bile

blood phlegm and black bile Hippocrates attributed the development of cancer to the

accumulation of black bile (Adams et al 1886) His theory of humorism was succeeded

by ideologies relating cancer to lymphatic dysfunction chronic irradiation trauma

inflammation and infectious disease The later thinkers were correct as recurrent

stressors radiation environmental exposures and viral infections underlie the etiology of

various cancer types In the 1800s however scientific inquiry transitioned from theories

seeking to purely identify the environmental contributions of cancer to a more cellular-

and molecular-driven understanding of cancer biology

With the advent of the microscope Johannes Muumlller also known as the first tumor

pathologist provided the first microscopic description of benign and malignant tumors in

1838 (Hajdu et al 2004) He was also the first to define malignancies as a collection of

individual cells Rudolph Virchow pathologist and student to Johannes Muumlller expanded

upon his findings by postulating that cancer arises from primitive undifferentiated cells

based on the histological similarities between tumors ad embryonic tissue (Huntly et al

2005) In 1875 Franco Durant and pathologist Julius Cohnheim introduced the

embryonal rest theory which links the origin of cancer to embryonic cells that remain in

a dormant state fail to mature during embryogenesis or fetal development and transform

1

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

into malignancies later in life (Huntly et al 2005) John Beard later proposed the

trophoblastic theory of cancer whereby cancer arises from aberrant germ cells that give

rise to multipotent or undifferentiated cancerous cells (Gurchot 1975) A common theme

underlying each of the aforementioned theories of the 19th and 20th centuries is the

common assignment of tumor origins to cells that harbor stem-like properties

The first definitive evidence for the existence of somatic stem cells occurred in 1963 by

studies conducted by Til and McCulloch in normal hematopoiesis In this landmark

article the authors heavily irradiated mouse bone marrow to produce cells with distinct

chromosomal aberrations and transplanted the genetically modified cells into irradiated

mice Within 10-14 days the mice had developed spleen nodules also known as colony-

forming unit spleen or CFU-Srsquos Each CFU-S was shown to possess a multilineage

potential and every cell within each colony contained the same aberrant karyotype

suggesting that the colonies had derived from a single cell (Becker et al 1963) In 1961

Southam and Brunschwig published an article on the transplantation of concentrated

cancer cell suspensions into patients with fatal disease Of the 27 patients studied only 6

had evidence of tumor growth The authors concluded that ge 1 million injected cancer

cells are required to initiate disease (Southam et al 1961) This study suggested that only

subsets of cells within a tumor are capable of engrafting disease It was therefore the

first to provide evidence for the heterogeneity of cellular function within a single tumor

Two competing theories originated from the 1961 and 1963 studies in attempt to explain

the heterogeneity of cancer cells The stochastic theory claims that tumors consist of

2

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

homogeneous cells with each cell having an equal opportunity to gain the capacity for

self-renewal and thus the ability to give rise to malignant growth In contrast the

hierarchical theory supported by findings of Til and McCulloch in normal

hematopoiesis defines malignancies as a collection of heterogeneous cells organized in a

hierarchical fashion According to this theory now known as the cancer stem cell (CSC)

theory each malignancy harbors a rare population of functionally and phenotypically

distinct CSCs that differentiate into bulk malignant cells making the isolation and

purification of CSCs a possibility (Wang et al 2005) John E Dick now considered one

of the pioneers of the CSC theory provided the first definitive experimental evidence for

this theory in 1994 The validation of this theory and the identification of potential driver

mutations have led to a better understanding of the origin of cancer and the causes of

therapeutic resistance disease progression and relapse

The purpose of my thesis is to explain our current understanding of malignant

transformation in acute myeloid leukemia (AML) and to describe how this knowledge

has aided the clinical assessment and treatment of this disease In order to demonstrate

this I will provide an introduction to the epidemiology and clinical manifestations of

AML the molecular mechanisms underlying its pathogenesis and new methods to risk-

stratify and treat the disease I will then provide a thorough discussion on normal

hematopoiesis and the cellular mechanisms of AML pathogenesis (in the context of the

CSC theory) and will conclude with a brief summary on a novel leukemic stem cell-

directed therapy that we are currently developing in our laboratory The study of cancer

3

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

has a long storied history For the first time in over 40 years however drastic changes

are underway in the way we evaluate and treat AML

4

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

2 Acute Myeloid Leukemia Background

21 Epidemiology

AML is a potentially life-threatening but rare clinical entity In 2013

14590 individuals were diagnosed with AML in the United States and

10370 patients died from the disease (Howlader et al 2013) The median

age of diagnosis is 67 years of age However AML is not necessarily a

disease of the elderly 174 of those diagnosed are lt 60 years of age

with children (0-19 years) and adolescents and young adults (AYAs 20-

39 years) accounting for 36 and 42 of the AML diagnoses made in

the US (Howlader et al 2013) (Figure 1) Individuals at an increased risk

of developing AML include those with certain congenital disorders (eg

Downrsquos syndrome neurofibromatosis type 1 congenital bone marrow

failure) and hematologic disorders (eg myelodysplastic syndrome

(MDS) myeloproliferative neoplasms) Furthermore individuals who

have been exposed to ionizing radiation benzene alkylating agents (eg

cyclophosphamide) or topoisomerase II inhibitors (eg etoposide) are also

at a significantly increased risk of developing this disease (Yagasaki et al

2009 Kreipe et al 2011 Jawad et al 2007 Khalade et al 2010 Cole et

al 2010) Interestingly alkylating agents and topoisomerase II inhibitors

used to treat other malignancies account for 10-15 of AML cases also

known as ldquotherapy-relatedrdquo or ldquosecondaryrdquo AML These patients

5

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

experience a very poor prognosis (2-year overall survival (OS) 8)

which is at least partially attributable to the poor prognostic karyotypes

(eg abnormalities in chromosomes 5 or 7) produced by DNA damaging

agents (Josting et al 2003 Kern et al 2004) Therefore changes in the

treatment of other malignancies including the eradication of conventional

chemotherapy could significantly reduce the incidence of AML alone

22 Clinical Manifestations

AML consists of a heterogeneous group of hematologic malignancies that

arise from immature hematopoietic cells It is characterized by the

presence of 20 myeloid blasts in the bone marrow (BM) with the

exception of t(1517) t(821) t(1616) and inv(16) which are diagnostic

of AML despite the blast percentage (Tallman et al 2005 NCCN)

Leukemic blasts exhibit various morphologic and immunophenotypic

traits that can be detected by immunohistochemistry and flow cytometry

These findings have led to the development of classification schemes that

reflect shared features among AML subtypes The French American

British (FAB) classification stratifies AML into eight groups M0-M7

Each group is named according to the degree of cellular maturation or the

immature cell type that has undergone clonal expansion Specifically M0-

M2 M3 M4 M5 M6 and M7 are characterized by the excess production

of malignant myeloblasts promyelocytes monoblasts monoblasts +

6

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

myeloblasts erythroid precursors +- myeloblasts and megakaryoblasts

(Arber et al 2003) (Table 1)

Regardless of the AML subtype however the accumulation of myeloid

blasts ensues as cytopenias or reductions in white blood cells (WBCs)

red blood cells (RBCs) and platelets Cytopenias have been attributed to

the physical crowding out of normal hematopoietic cells by malignant

blasts However recent studies suggest that the activation of aberrant

molecular pathways and the production of excess cytokines by leukemic

cells suppress hematopoiesis (Kats et al 2014 Miraki-Moud et al 2013

Buggins et al 2001) Cytopenias clinically manifest in the form of fatigue

excess bleedingbruising and increased risk of infection and are the

primary mediators of patient demise Without treatment patients die of

bleeding or infectious complications within weeks Current therapeutic

interventions are effective in obliterating bulk leukemic blasts and thus

reversing the cytopenias for months-to-years however most patients

eventually relapse with overwhelming infections being the most common

cause of death (Tallman et al 2005)

23 Pathogenesis

Various cytogenetic abnormalities and molecular lesions have been

identified in AML however no single driver mutation has been

7

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

recognized This indicates that AML has numerous molecular origins and

requires cooperating oncogenic events for transformation The two-hit

hypothesis a widely accepted model for AML provides an explanation

for these findings According to this model a combination of pro-

proliferativepro-survival mutations (ldquoclass Irdquo eg Ras c-Kit FLT3) and

differentiation blocking mutations (ldquoclass IIrdquo eg CBFβ-MYH11 inv(16)

AML1-ETO PML-RARα AML1 TEL-AML1) are required for

transformation of normal hematopoietic stemprogenitors into leukemic

cells (Gilliland et al 2001 Reilly et al 2004) Evidence in support of this

model stems from the observed latency of transformation in patients and

murine models harboring germline leukemic mutations correlative studies

linking class I and class II mutations with the development of AML and

in vivo functional assays that have further validated these correlative

studies

The extended time to leukemic transformation has been observed in

patients with familial platelet disorder (FPD) and murine models of AML

FPD is a disease that is characterized by the germline inheritance of a

heterozygous mutation in RUNX1 (Owen et al 2008) This mutation

increases the risk of myeloid malignancies particularly AML and MDS

Despite the presence of the germline mutation only 35 of patients

develop AML and the median age of diagnosis is gt65 years (Owen et al

2008 Office for National Statistics 2004) Similarly studies in mice

8

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

aberrantly expressing the PML-RARα translocation have been shown to

develop AML with a median latency of 85 months and an incomplete

penetrance of 64 (Brown et al 1999) Lastly transgenic mice expressing

PML-RARα and BCL-2 have been shown to develop AML with a median

latency of 127 days (Kogan et al 2001) The long-latency and incomplete

penetrance suggests that additional mutations or karyotypic abnormalities

are required for transformation

Consistent with this data Kats et al (2014) found that mice expressing

mutant IDH2 fail to develop a leukemic phenotype in the absence of

additional genetic (eg class I FLT3-ITD) or non-genetic alterations (eg

HoxA9Meis1 overexpression) Similarly the concurrent expression of

class I FLT3-ITD with class II AML1 mutations or class II PML-RARα

translocations robustly transform hematopoietic stemprogenitor cells into

AML (Kottaridis et al 2001 Kelly et al 2002a Matsuno et al 2003)

while the expression of class I mutant FLT3-ITD alone leads to the

development of myeloproliferative disorders (MPD) not AML (Kelly et

al 2002b Lee et al 2005) Furthermore mice transfected with class II

CBFβ-MYH11 or AML1-ETO fail to develop AML in the absence of

additional mutations (Castilla et al 1996 Kogan et al 1998 Castilla et al

1999 Rhoades et al 2000 Higuchi et al 2002) Therefore class I and

class II mutations seem to be necessary for leukemogenesis

9

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Despite such evidence the two-hit hypothesis has been recently

challenged Mutations in epigenetic modifiers account for gt 49 of the

molecular aberrations observed in AML These mutations are critical to

leukemogenesis and occur in combination with class I and class II

mutations (Patel et al 2012 Shih et al 2012) Therefore many have

suggested that the original model be revised to include class III mutations

in epigenetic modifiers (Greenblatt et al 2014 Shih et al 2012) Class III

mutations can be further subdivided into two distinct categories - those

that affect DNA hydroxymethylation (eg IDH1 IDH2 and TET2) and

those that directly or indirectly regulate DNA methylation (eg MLL

translocations and mutations in ASXL1 and DNMT3A) (Shih et al 2014)

In addition changes in the BM microenvironment microRNAs and non-

genetic alterations in epigenetic modifiers have been shown to mimic the

biologic effects of mutated leukemogenic genes These findings challenge

the notion that two distinct genetic lesions are required for transformation

A landmark article by Kode et al (2014) found that β-catenin activating

mutations in mouse osteoblasts mediate robust transformation into AML

and give rise to mutations and chromosomal aberrations observed in the

clinic (Kode et al 2014) In another study Shultz and colleagues found

that PML-RARα is insufficient for leukemogenesis but that IL-3 signaling

cooperates with PML-RARα to mediate transformation (Shultz et al

2000) The pro-proliferative function of IL-3 suggests that it may be

categorized as a non-genetically modified class I molecule thus

10

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

explaining why the PML-RARαIL-3 combination mediates

transformation In another study Han et al (2010) found that

overexpressing microRNA-29a in early hematopoietic cells stimulates

robust transformation into AML in the absence of additional engineered

mutations This microRNA has been shown to promote cellular

proliferation and to block myeloid differentiation therefore microRNA-

29a may act as a non-genetically modified class I and class II microRNA

during AML pathogenesis (Garzon et al 2009 Wang et al 2012 Gu et al

2013 Qin et al 2011) Similarly microRNA-125b is strongly up-

regulated in a rare form of AML and its overexpression has been shown

to block monocytic and granulocytic differentiation in cell lines

(Mousquet et al 2008 Klusmann et al 2010) Lastly altered TET2 and

DNMT3A activity has been detected in patients harboring wild-type TET2

and DNMT3A (Ko et al 2010) Changes in microRNA expression (eg

microRNA-29 targeting of TET2 and DNMT3A) andor co-occurring

mutations (evidenced by altered TET2 activity in IDH mutant AML) may

be responsible for these findings (Cheng et al 2013 Garzon et al 2009

Shih et al 2012) Thus incorporating class III mutations mutations in the

BM microenvironment and the disease-modifying effects of non-genetic

changes into the two-hit hypothesis are critical to understanding the true

mechanisms underlying leukemogenesis

23 Implications of Molecular Aberrations on Clinical Decisions

11

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

After being diagnosed with AML patients are stratified into three risk

categories favorable (5-yr OS 34-55) intermediate (5-yr OS 13-38)

and unfavorable disease (5-yr OS 2-11) (Byrd et al 2002 Slovak et al

2000 Grimwade et al 2001) Until 2008 cytogenetics and dysplasia were

used to prognosticate patients into the different risk categories (NCCN)

However ~50 of patients with AML have normal cytogenetics

(cytogenetically normal AML CN-AML) (Grimwade et al 2001) The

high proportion of CN-AML and the inability to accurately risk stratify

this patient population led to the widespread utilization of whole-genome

and exome sequencing to identify recurrent somatic mutations that could

risk stratify patients with CN-AML The outcomes of these efforts in

combination with sequencing efforts in patients harboring karyotypic

abnormalities have led to the incorporation of molecular diagnostics in

the risk stratification of AML (Table 2)

According to the NCCN guidelines CN-AML is categorized as an

intermediate-risk disease that has favorable outcomes in the presence of

NPM1 or CEBPα mutations and poorer outcomes in the presence of

FLT3-ITD mutations (NCCN) In addition core-binding factor AML

(CBF AML) which includes inv(16) and t(821) carry a favorable

prognosis In the presence of c-Kit mutations however CBF-AML

becomes an intermediate-risk disease (NCCN) Additional studies on

CEBPα found that double allelic mutations at the CEBPα locus serve as a

12

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

favorable prognostic marker while outcomes of single CEBPα mutant

AML are similar to wild-type AML (Taskesen et al 2011 Pabst et al

2009) Therefore distinguishing single and double allelic mutations in

CEBPα could be critical for the accurate risk stratification of patients

In a study by Patel et al (2012) 398 patients with intermediate-risk

disease (eg CN-AML) enrolled in the Eastern Cooperative Oncology

Group (ECOG) clinical trial were further risk stratified based on the

presence of additional molecular aberrations (Table 3) CN-AML with

mutated NPM1 in the absence of FLT3-ITD previously categorized as

good-risk AML was shown to carry an extremely favorable outcome

(greater than that of CBF AML) in the presence of IDH1 or IDH2

mutations Intermediate-risk AML patients with wild-type FLT3-ITD

carried a worse prognosis in the presence of TET2 MLL-PTD ASXL1 or

PHF6 mutations Intermediate-risk AML patients without these mutations

experienced improved outcomes relative to intermediate-risk AML with

mutant CEBPα Furthermore intermediate-risk AML carrying the FLT3-

ITD allele was associated with the worst prognosis among intermediate-

risk patients in the presence of TET2 MLL-PTD DNMT3 mutations or

trisomy 8

Despite these promising conclusions conflicting results have been

reported on the prognostic implication of IDH and CEBPα mutations In

13

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

contrast to the findings by Patel and colleagues (2012) Ravandi et al

(2012) found that IDH mutations confer a worse prognosis in NPM1

mutant AML Furthermore Ravandi et al (2012) showed that isolated

IDH mutations carry no prognostic significance In contrast Koszarska et

al (2013) demonstrated that IDH1 R132H confers a worse prognosis

relative to IDH wild-type or IDH R132C and that specific amino acid

changes influence clinical characteristics and disease outcomes While

Patel et al (2012) demonstrated that mutant CEBPα presents an

intermediate-risk disease despite FLT3-ITD mutations Taskesen et al

(2011) described mutations in NPM1 and FLT3-ITD as being dominant

over CEBPα mutations These findings highlight the importance of taking

multiple co-occurring mutations and specified mutational subtypes into

account in prognosticating patients and likely accounts for much of the

unexpected variability in clinical outcomes observed using current risk

stratification guidelines

While sequencing technology has created a platform for the identification

of genetic alterations monosomal karyotypes (MKs) and Wilmrsquos tumor

gene (WT1) are also being investigated as potential prognostic markers

MKs have been identified as very poor prognostic markers (4-year

survival 9-12) particularly in those with ge2 monosomies or one

monosomy with an additional structural abnormality The latter has been

shown to confer an even worse prognosis (4-year OS lt4) (Kayser et al

14

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

2012 Breems et al 2008 Medeiros et al 2010) MK patients are typically

older and lack NPM1 or FLT3 mutations Their poor prognosis is partially

attributed to the high correlation between MKs and p53 mutations (Kayser

et al 2012 Rucker et al 2011) Similarly the up-regulation of WT1 has

been detected in AML and is associated with a significant reduction in

rates of complete remission (CR) disease-free survival (DFS) and OS

especially when combined with other molecular risk factors (Ziaodong et

al 2014) Studies are currently evaluating whether WT1 mRNA can be

leveraged as a marker of relapse following treatment (Yamauchi et al

2013)

24 Targeting Recurrent MolecularChromosomal Aberrations

The treatment of AML has remained relatively unchanged within the past

40 years The therapeutic approach typically includes a series of induction

consolidation and maintenance therapies The lsquo3+7rsquo regimen is used for

induction with cytarabine a cytosine nucleoside analog continuously

administered for seven days intravenously and an anthracycline or DNA

intercalator (eg daunorubicin or idarubicin) administered at a single

intravenous dose for the first three days of treatment (Fernandez et al

2010) The goal of induction therapy is to achieve CR however the lsquo7+3

regimenrsquo yields a short-lived CR in the absence of post-remission therapy

Therefore patients are risk stratified for consolidation patients with

15

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

unfavorable risk disease are typically treated with hematopoietic stem cell

transplantation (HSCT) or entered into clinical trials while patients with

favorable molecular studies and cytogenetics are treated with high-dose

cytarabine (HDAC) for consolidation

While these therapeutic strategies are beneficial in delaying hematopoietic

failure and prolonging survival relapse remains a significant challenge in

the treatment of AML nearly all patients succumb to their disease HSCT

is the only potentially curative strategy (Beckerich et al 2013) However

only a limited number of patients are eligible for transplantation and

HSCT is not effective in all patients Eligibility is determined by various

factors including age comorbidities blast percent and the presence of

HLA-matched siblings Duval et al (2010) also demonstrated the value of

using the duration of CR favorably cytogenetics presence of HLA-

matched related donors KarnofskyLansky scores and the absence of

circulating blasts to develop a scoring system that would identify patients

who would benefit from HSCT According to this system patients with

scores of 0 1 2 and 3 have a ~45 30 15 and 6 chance of

survival 3-years following transplantation (Duval et al 2010) Therefore

HSCT is effective in some but not all patients Moreover patients treated

with HSCT are at a high risk of treatment-related mortality (~30-40) and

of developing complications of transplantation (eg graft-versus-host-

disease) (Litzow et al 2010 Tallman et al 2000) As a result clinicians

16

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

researchers and pharmaceutical companies have sought to identify novel

therapeutic targets and personalized agents that would improve patient

outcomes Pharmacologic and biologic inhibitors or modulators of

DNMT3ATET2 FLT3-ITD IDH1 IDH2 MLL fusions and WT1 are

currently under investigation for the treatment of AML

DNMT3A and TET2 are epigenetic modifiers that are mutated in 23 and

8 of AML patients Mutations in each of these molecules confer a poor

prognosis (Ley et al 2010 Thol et al 2011 Patel et al 2012) DNMT3A

is a DNA methyltransferase that functions in the methylation of genomic

CpG dinucleotides However in vivo deletions of DNMT3A mediate both

the hypermethylation and hypomethylation of various loci (Shih et al

2012) Hypomethylation occurs on the promoter of genes critical to HSC

self-renewal consistent with data indicating the expansion of long-term

HSCs in DNMT3-null xenotransplantation models (Shih et al 2012) In

contrast TET2 is an enzyme responsible for catalyzing the conversion of

5-methylcytosine to 5-hydroxymethylcytosine a method of DNA

demethylation TET2-null mice have been shown to develop

myeloproliferative diseases suggesting that TET2 serves as a tumor

suppressor (Moran-Crusio et al 2011 Pronier et al 2011 Ko et al 2011)

This finding is consistent with TET2 expression in HSCs and its role in

self-renewal lineage commitment and terminal differentiation (Solary et

al 2014) Hypomethylating agents (HPAs including decitabine and

17

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

azacitidine) are currently FDA-approved for the treatment of MDS but

not AML However HPAs have been shown to alter the clinical course of

TET2 mutant AML and to directly target DNMTs (Itzykson et al 2011

Hagemann et al 2011) They are effective in the treatment of elderly

patients with AML who are ineligible for standard induction

chemotherapy and in AML patients harboring TET2 or DNMT3A

mutations (independent of adverse cytogenetics) (Cashen et al 2010

Itzykson et al 2011 Metzeler et al 2012) Large-scale prospective studies

are necessary to validate the clinical utility of HPAs in AML

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase that is mutated or

acquires an internal-tandem duplication (ITD) in 37 of AML patients

(Patel et al 2012) Upon activation FLT3-ITD dimerizes undergoes auto-

phosphorylation and activates downstream pro-proliferativepro-survival

signaling pathways (Marchetto et al 1999 Dosil et al 1993 Scheijen et

al 2004) In normal hematopoiesis FLT3 controls the growth and

differentiation of immature hematopoietic cells (Stacchini et al 1996) In

malignant hematopoiesis however FLT3-ITD stimulates proliferation and

blocks myeloid differentiation (Hirade et al 2013) Despite this FLT3

mutations are insufficient for the development of AML In the presence of

additional cytogenetic aberrations however mice have been shown to

develop AML with a short latency and 100 penetrance Sorafenib a

FLT3 inhibitor has been shown to reduce the leukemic burden almost

18

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

completely in the peripheral blood mononuclear cells (PBMNCs) of mice

expressing FLT3-ITD mutant AML 4 weeks following the initiation of

treatment (Greenblatt et al 2012) Sorafenib has also been shown to

induce growth arrest and apoptosis in vitro and to reduce leukemic burden

and prolong survival in mice (Zhang et al 2008) To date FLT3

inhibitors lestaurtinib midostaurin sorafeinib quizartinib have been

tested in Phase III clinical trials The survival benefit of these studies

have been disappointing thus far and may be attributed to the fact that

FLT3 mutations are late events in the clonal evolution of cancer (as

described later in this review) Nevertheless quizartinib the most potent

of the FLT3 inhibitors has been shown to mediate significant reductions

in BM blasts in the absence of systemic chemotherapy and is therefore

beneficial in preparing FLT3-ITD mutant AML patients for HSCT (Levis

2013) FLT3 inhibitors are also being tested for efficacy in combination

with standard chemotherapy Therefore FLT3 inhibitors may enter the

clinic in the near future

IDH1 and IDH2 are metabolic enzymes mutated in 15-33 of AML

patients (Shih et al 2012) The IDH family is responsible for catalyzing

the conversion of isocitrate into α-ketoglutarate in the Krebrsquos cycle

However gain-of-function mutations in IDH1 and IDH2 promote the

conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG) which has

been shown to block myeloid differentiation (Shih et al 2012) Mutant

19

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

IDH2 leads to extramedullary hematopoiesis and the expansion of early

hematopoietic cells (eg stem cells and progenitors) in mouse xenograft

models while removing the IDH2 mutant has been shown to reverse

IDH2-mediated leukemic phenotypes (Kats et al 2014) Genetic knock-in

mutations of IDH1 R132H the most common IDH1 mutation increase the

numbers of early hematopoietic progenitors and lead to the development

of splenomegaly anemia and extramedullary hematopoiesis in murine

models (Sasaki et al 2012) Importantly pharmacologic inhibition of 2-

HG robustly reverses the myeloid differentiation blockade (Thompson et

al 2013 Kats et al 2014) Clinical trials targeting IDH are currently

underway An interim analysis from the Phase I IDH2 inhibitor trial in

patients with relapsedrefractory AML has demonstrated an objective

response in 610 patients treated with the IDH2 inhibitor 2 patients are in

CR 3 are in CR with incomplete platelet recovery and 1 is in partial

remission Consistent with the preclinical studies on IDH inhibition 2-HG

levels decrease by up to 97 in patients and myeloblast differentiation is

profoundly stimulated (AACR) The outcomes of the IDH2 inhibitor are

promising but also suggest that 2-HG may be a valuable biomarker for

response to IDH directed therapies Recruitment for Phase I IDH1 clinical

trials began in March 2014

The MLL gene encodes an epigenetic modifier that is frequently mutated

in AML and accounts for 14-22 of the molecular and cytogenetic

20

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

aberrations observed in AML (Shih et al 2012) MLL-translocations

produce in fusion proteins including MLL-AF4 -AF9 -AF10 (or ndashENL)

and ndashAF6 which account for 80 of these rearrangements The MLL

fusions activate self-renewal in committed hematopoietic cells and

mediate leukemogenesis in various in vitro and in vivo assays (Krivtsov et

al 2007) These phenotypes are believed to stem from the interaction

between MLL-fusion partners and DOT1L a histone methyltransferase

that promotes gene expression (Okada et al 2005 Zhang et al 2006

Bitoun et al 2007 Zeisig et al 2005 Erfurth et al 2004) siRNA-

mediated knock down of DOT1L abrogates leukemic transformation by

MLL-AF10 and depleting DOT1L eradicates MLL-AF9 leukemic cells

(Okada et al 2005 Nguyen et al 2011) These effects are at least partially

attributed to the DOT1L regulation of Hoxa and Meis1 (Nguyen et al

2011) Clinical trials targeting DOT1L are currently underway Results

from the Phase I studies have shown its efficacy in stimulating myeloid

differentiation and in reducing blast counts Expansion of the Phase I

clinical trial began in December 2013

WT1 encodes a transcription factor that is known for its role in the

development of childhood renal carcinoma However it is also highly

expressed at diagnosis up-regulated during relapse and mutated in 8 of

AML patients WT1 has been shown to promote cellular proliferation and

to block differentiation in AML (Inoue et al 1998) and antibodies (Abs)

21

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

targeting WT1 have demonstrated a significant efficacy in vivo These Abs

reduce BM blast percentages to lt5 10 weeks post-treatment and leads to

CR 16 weeks following treatment initiation (Mailander et al 2004) In one

study 510 patients went into or were maintained in CR Notably 5 of the

5 patients who responded were expected to experience relapse in the

absence of the vaccine In another Phase I study 55 AML patients

experienced reductions in minimal residual disease (MRD) and a long-

lasting CR (Oka et al 2004) In a case report of 3 AML patients the WT1

vaccine led to long-lasting complete remissions of gt7-9 years in all

patients Stopping the treatment led to elevations in WT1 mRNA

suggestive of disease progression and re-initiating treatment suppressed

WT1 mRNA levels back to basal levels Therefore the WT1 vaccine

seems to suppress malignant growth and to serve as an effective agent for

maintenance therapy WT1 mRNA may also serve as an effective

biomarker for MRD and disease progression in WT1 expressing AML

(Tsuboi et al 2012) Clinical trials on WT1 vaccinations are currently

ongoing and clinical trials targeting WT1 by chimeric-antigen receptor

(CAR) therapy are currently underway in the UK

22

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

3 Normal hematopoiesis

31 Hierarchical Organization of the Hematopoietic System

The hematopoietic system is organized in a hierarchical manner whereby

rare quiescent self-renewing HSCs give rise to downstream progenitors

(transit-amplifying cells) and terminally differentiated cells In this

model HSCs maintain myeloid and lymphoid homeostasis all throughout

life through their capacity to undergo symmetric and asymmetric divisions

(Figure 2) Mature hematopoietic cells are capable of giving rise to more

differentiated cells yet with a concomitant inability to de-differentiate

(Doulatov et al 2012) (Figure 3) Multipotent progenitors (MPPs Lin-

CD34+CD38-CD90-CD45RA-) arise from early differentiation events

downstream HSCs (Lin-CD34+CD38-CD90+CD45RA-) (Majeti et al

2007) MPPs are similar to HSCs in that they retain multilineage potential

however they possess a limited self-renewal capacity Consequently

MPPs are responsible for the rapid short-term reconstitution of blood

lineages in myeloablated patients while HSCs provide slower long-term

reconstitution The MPPs then divide and give rise to transit-amplifying

progenitors with variable proliferative potentials Common lymphoid

progenitors (CLPs CD34+CD38+CD10+CD19-) differentiate into natural

killer cells (NK Lin+CD34-CD38+CD56+) T lymphocytes (Lin+CD34-

CD38+CD3+) and B lymphocytes (Lin+CD34-CD38+CD19+) whereas

23

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

common myeloid progenitors (CMPs Lin-CD34+CD38+CD123+CD45RA-)

differentiate into granulocyte-macrophage progenitors (GMPs Lin-

CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors

(MEPs Lin-CD34+CD38-CD123loCD45RA-) that terminally differentiate

into granulocytes (eg neutrophils) (CD15+) monocytes (CD14+)

megakaryocytes (CD41+) and erythrocytes (red blood cells RBCs

GPACD235a+) GMPs have a high proliferative potential relative to

CLPs This difference is partially attributed to the functional differences in

these cell types While B and T lymphocytes proliferate and differentiate

in the periphery granulocytes and monocytes are shorter-lived cell types

that are dependent on the BM for consistent regeneration More recently

multilymphoid progenitors (MLP Lin-CD34+CD38-CD45RA+CD90-) also

known as lymphoid-primed multipotential progenitors (LMPPs) have

been identified as cells with the capacity to differentiate into both myeloid

(GMP) and lymphoid (NKTB) cells (Doulatov et al 2012)

The hierarchical organization of the hematopoietic system presents several

adaptive advantages First the low proliferative index and thus low

metabolic demands of HSCs protect them from acquiring mutations

through errors in DNA replication and oxidative stress Second despite

the relatively quiescent state of stem cells HSCs possess a large potential

for hematopoietic expansion that is primarily manifested through the high

proliferation of their daughter cells This property allows the rapid

24

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

amplification of blood cells as needed Third proliferating cells (eg

transit-amplifying cells) have a greater probability of acquiring random

mutations however these mutations are lost in the gene pool given the

limited lifespan of these cells and their inability to self-renew Lastly

blood cell synthesis is highly specific given the tight regulation of the

hematopoietic system Hemolysis of RBCs for instance leads to anemia

which stimulates the release of erythropoietin from the renal

juxtaglomerular apparatus and the proliferation of erythroid precursors in

the BM (Hăulică et al 1985) Similarly acute bacterial infections lead to

increased production and release of granulocyte-colony stimulating factor

(G-CSF) which stimulates the production of neutrophils (Selig et al

1995) Nevertheless the hematopoietic system is complex and the

theoretical advantages of this system do not necessarily equate with the

observed findings that lead to malignant hematopoiesis

32 HSC Immunophenotyping

Multi-parameter fluorescence-activated cell sorting (FACS) has played a

critical role in elucidating the cellular organization of hematopoiesis This

method allows the prospective isolation of antibody-labeled cells based on

distinct immunophenotypes and makes it possible to assay purified cells

for self-renewal and multilineage potentiality Classically self-renewal

has been determined in vitro using long-term culture initiating cell (LTC-

25

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

IC) assays and in vivo by serial transplantation and limiting dilution

analyses (LDA) (Sieburg et al 2002) Moreover multilineage potentiality

has been tested through the ability of single cells to give rise to various

cell lineages in vitro via methylcellulose assays and in vivo via

transplantation The greater a cells ability to serially plate and give rise to

lymphomyeloid grafts upon transplantation and the greater its multilineage

potential the higher its position in the hematopoietic hierarchy Using

these techniques the CD34+CD38- immunophenotype has been shown to

mark cells of early hematopoiesis including HSCs and early progenitors

(eg CMP CLP GMP) while CD34+-CD38+ marks more differentiated

rapidly proliferating cells (Hao et al 1995) Furthermore functional

analyses of various immunophenotypic cell compartments have

characterized HSCs as Lin-CD34+CD38-c-Kit(CD117)+CD90(Thy-

1)+CD45f+rhodaminelo expressing cells (Krause et al 1994 Okada et al

1992 Fleming et al 1993 Notta et al 2011) Despite this widely accepted

HSC phenotype no single multi-parameter cell surface marker analysis

has been able to identify pure populations of HSCs in humans

The difficulty of identifying individual HSCs is illustrated by recent work

from our laboratory While HSCs are widely accepted as c-Kit+ cells our

laboratory recently identified distinct subsets of HSCs based on c-Kit

expression (Shin et al 2014) According to this study low c-Kit

expressing HSCs (c-Kitlo HSCs) display increased self-renewal and long-

26

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

term reconstitution relative to high c-Kit expressing HSCs (c-Kithi HSCs)

c-Kithi HSCs have a megakaryocytic lineage bias while c-Kitlo HSCs are

not biased towards any particular lineage lastly c-Kithi HSCs arise from

c-Kitlo HSCs while the reverse does not hold true These findings suggest

that c-Kit expression could be used to isolate two distinct HSC

populations with c-Kitlo HSCs sitting at the apex of the hierarchy and c-

Kithi HSCs situated downstream

The challenge of using distinct sets of markers to isolate HSCs is also

evidenced by the presence of HSCs in CD34- cell populations In 1996

Osawa et al demonstrated the presence of HSCs in CD34-lo fractions

They showed that CD34-lo HSCs are capable of engrafting secondary

recipients and of giving rise to CD34+ cells in xenografted models (Osawa

et al 1996) Studies by Bhatia et al (1998) and Zanjani et al (1998)

found that Lin-CD34- cells isolated from human cord blood show long-

term activity in vitro and long-term multilineage reconstitution upon

transplantation into NODSCID mice with CD34- cells giving rise to

CD34+ progenitors In sheep Lin-CD34- and Lin-CD34+ human BM cells

are able to initiate long-term grafts with multilineage differentiation upon

transplantation and are capable of giving rise to one another suggesting

that the CD34 phenotype may be reversible Taken together distinct HSCs

exist that possess differences in self-renewal lineage bias and quiescence

and are organized in a hierarchical fashion that may rely on reversible

27

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

phenotypes Nevertheless CD34+ expression has been used widely

accepted as the HSC immunophenotype given the higher frequency of

HSCs within this compartment (140-44 Lin-CD34+CD38- 11000 Lin-

CD34-) (Yahata et al 2003 Ishii et al 2011)

4 AML and the Cancer Stem Cell Theory

28

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

41 Proof of LSCs

In 1994 a landmark article published by John Dick and colleagues

provided the first definitive evidence for the presence of CSCs in cancer

In this study AML cells were fractionated into CD34+CD38- and

CD34+CD38+ populations by FACS transplanted into SCID mice and

evaluated for stem-cell properties such as AML reconstitution serial

transplantability and the ability to recapitulate features of the original

AML The authors found that immunophenotypically immature

CD34+CD38- cells constitute 1-1 of the AML population and contain a

SCID-leukemia initiating cell (SL-IC) frequency of 1250000 cells while

neither CD34+CD38+ nor CD34- populations contain SL-ICs (Lapidot et al

1994) However it was not until the development of NODSCID mice

which harbor deficiencies in both innate and adaptive immunity when SL-

ICs were validated as LSCs The reduced immunodeficiency of these mice

enhanced the purification of HSCs by 10-20-fold thereby allowing serial

transplantations and providing evidence for the self-renewal capacity and

reconstitution of AML by these cells

Increasingly immunodeficient mice including

NODSCIDβ2microglobulin-- mice and NODSCIDIL2Rγ-- (NSG) mice

further improved engraftment and enhanced our understanding of the LSC

29

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

immunophenotype in AML (Christianson et al 1997 Shultz et al 2005)

The low frequency of SL-ICs in the Lapidot et al study (1250000 cells)

for instance suggested that either CD34+CD38- populations contain LSCs

but at a low frequency or that engraftment is diminished by the active

immune surveillance of recipient mice Ishikawa et al (2007) found that

the degree of immunodeficiency of mouse recipients play a major role in

determining engraftment efficiency They showed that transplanting 1000

CD34+CD38- cells engrafted AML 250-fold more in NSG mice relative to

the number used in the Lapidot et al study However transplanting cell

numbers as high as gt 106 CD34+CD38+ and CD34- cells failed to engraft

AML supporting the findings of the original study

Similar to the immunophenotyping of HSCs however the LSC

immunophenotype is still being investigated In one study the anti-CD38

antibodies (Abs) used in xenotransplantation assays were shown to inhibit

the engraftment of CD34+CD38+ leukemic cells through an Fc receptor-

dependent mechanism (Taussig et al 2008) The authors found that pre-

treating mice with intravenous immunoglobulin (IVIG) followed by anti-

CD38 Ab infusions blocked the Fc-receptors and improved engraftment

This led to the discovery of SL-IC activity in the Lin-CD34+CD38+

compartment and the absence of LSC activity in CD34+CD38- cells of

some primary AML patient samples (Taussig et al 2008) In another

study NPM1 mutant AML samples were split into two groups patient

30

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

samples that consist of lt05 CD34+ cells (group A) and those that

possess gt05 CD34+ cells (group BC) Group BC were shown to

possess LSC activity in both the CD34+ and CD34- fractions while LSCs

were restricted to the CD34- compartment in Group A (Taussig et al

2010) Interestingly the CD34+ cells of group BC were shown to

recapitulate AML with CD34- dominance upon transplantation (Martelli et

al 2010 Taussig et al 2010) These findings are attributable to the CD34-

dominance of the NPM1 mutant AML subtype the use of IVIG pre-

treatment in severely immunodeficient NODSCIDβ2microglobulin-- and

NSG mice or both The authors also found that CD34+ engrafting LSCs

fail to express CD34 in vivo suggesting that CD34+ cells lose their CD34

expression upon transplantation Therefore CD34+ and CD34- LSCs may

be organized in a hierarchical fashion Alternatively the CD34 phenotype

may be reversible Nevertheless these findings clearly indicate the

heterogeneity of LSC immunophenotypes in NPM1 mutant AML and the

presence of LSCs in the CD34+CD38+ compartment in some AML patient

samples

While the LSC immunophenotype seems to be relatively inconsistent

CD34+CD38- is a widely accepted LSC phenotype Sarry et al (2011)

shed light on the accuracy of this phenotype in isolating LSCs In this

study the authors sought to functionally validate the presence of LSCs in

various immunophenotypic compartments (Lin+- CD34+- CD38+-) within

31

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

individual AML patient samples and found that all phenotypes examined

are capable of engrafting AML in NSG mice However LSCs were highly

enriched in the CD34+CD38- compartment of NPM1 wild-type AML

Therefore the CD34+CD38- phenotype represents the greatest fraction of

LSCs in NPM1 wild-type AML

These studies confirm the immunophenotypic heterogeneity of LSCs

within both individual patient samples and across various AML samples

They also emphasize the importance of utilizing severely immunodeficient

murine models in accurately identifying LSCs

42 LSC Cell of Origin

The cell of origin in AML has remained controversial with some groups

supporting the transformation of HSCs or MPPs into LSCs and other

suggesting that downstream progenitors serve as AML LSCs Two major

arguments have been made in support of the former First the

immunophenotypic similarities among LSCs and HSCs (both Lin-

CD34+CD38-) suggest that HSCs are the cell of origin (Bonnet et al

1997) Similarly Majeti et al (2007) argued that the CD90- phenotype of

LSCs point to Lin-CD34+CD38-CD90- MPPs also known as short-term

HSCs as the cell of origin Second self-renewal is an intrinsic property

that is required to initiate and propagate AML and this feature is absent in

32

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

progenitors Therefore some claim that it is unlikely that progenitors are

mediators of transformation unless the earliest mutations provide aberrant

self-renewal capabilities (Wang et al 2005) Since HSCs are long-lived

however they are arguably the prime candidates for transformation (Wang

et al 2005) Despite these claims downstream progenitors have been

functionally validated as the LSCs or leukemia initiating cells in AML

Mice transplanted with myeloid progenitors (eg GMPs) aberrantly

expressing MLL-AF9 or MLL-ENL have been shown to develop AML

(Krivtsov et al 2006 Cozzio et al 2003) The gene expression profiling

of LSCs from these mice showed that they were GMPs that had aberrantly

activated genes related to stemness (eg Hoxa9 Hoxa10) also known as

GMP-like cells (Kristov et al 2006) In another study hematopoietic

stemprogenitor cells stably expressing microRNA-29a were transplanted

into lethally irradiated mice The mice developed a myeloproliferative

disease characterized by GMP and in some cases CMP expansions

which progressed to AML (Han et al 2010) Importantly the GMPs and

CMPs were found to acquire aberrant self-renewal capabilities evidenced

by their ability to give rise to long-term grafts and by serial dilution

analyses that showed a very high LSC frequency (120) within the GMP

compartment The most robust evidence pointing to downstream

progenitors as the origin of LSCs however stems from a study by

Goardan et al (2011) in CD34+ AML The authors identified the

33

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

expansion of CD34+CD38- LMPP-like cells and CD34+CD38+ GMP-like

cells in 80 of AML patient samples They also found that the LMPP- and

GMP-like cells co-exist possess LSC activity and exhibit distinct

molecular profiles Moreover the LMPP-like cells were shown to have a

higher LSC frequency greater self-renewal potential and the ability to

differentiate into CD34+CD38+ GMP-like LSCs In contrast the GMP-like

cells were unable to differentiate into LMPP-like LSCs Furthermore

LMPP-like LSCs were found to be enriched for genes that are up-

regulated in immature FAB AML subtypes (M0 and M1) while GMP-like

LSCs were enriched for genes up-regulated in more mature AML subtypes

(M2 M4 and M5) Consequently the authors concluded that LMPP-like

LSCs are situated at the apex of the hierarchy in AML development The

results from this study are supported by previous conclusions by Ishikawa

et al (2007) wherein CD38-CD45RA+ LSCs were found to lie at the apex

of the AML hierarchy and by Eppert et al (2011) who identified a

greater LSC frequency in Lin-CD34+CD38- cells relative to CD34+CD38+

cells These studies strongly suggest that LMPP- and GMP-like cells are

AML LSCs that arise from downstream progenitors (LMPPs GMPs)

43 Current Model for the Hierarchical Organization of AML

While the final transforming event in AML seems to occur in LMPPs

GMPs andor CMPs studies have shown the presence of co-existing

34

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

molecular aberrations in leukemic blasts and mature lymphoid cells These

findings suggest that cells (eg MPPs HSCs or LMPPs) upstream the

myeloid committed progenitors serve as a reservoir of preleukemic cells

The first evidence for the presence of preleukemic HSCs stemmed from a

study in patient samples harboring FLT3-ITD mutant CN-AML In this

study residual HSCs were isolated from 6 patient samples using the Lin-

CD34+CD38-TIM3-CD99- markers and were transplanted into NSG mice

(Jan et al 2012) The residual HSCs engrafted with multilineage

potentiality (CD33+ myeloid and CD19+ lymphoid cells) they also lacked

the FLT3-ITD mutation suggesting that the HSCs were non-malignant

cells Despite this 32 of the 51 mutations analyzed were found in the

residual HSCs many of which were present in the leukemic blasts In

addition each of the patient samples acquired FLT3-ITD as a late event

The authors concluded that residual HSCs represent a small reservoir of

preleukemic cells that harbor founder mutations are capable of normal

differentiation but lack the complete complement of mutations required

for transformation into overt AML This study also identified FLT3-ITD

as a common late event in the clonal evolution of AML

Preleukemic HSCs have also been identified in t(821) (AML1ETO) as

well as NPM1c DNMT3A and IDH2 mutant AML (Miyamoto et al

2000 Shlush et al 2014) In a study by Shlush et al (2014) the authors

studied AML samples harboring both DNMT3A and NPM1c mutations in

35

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

order to use NPM1c as a marker for preleukemic cells given the

acquisition of DNMT3A mutations as late events Consistent with their

hypothesis the authors found that preleukemic cells harbor mutations in

NPM1c while co-occurring NPM1cDNMT3A mutations are only present

in GMPs andor LMPPs and leukemic blasts These findings point to

GMPs andor LMPPs as the cells of origin for transformation further

supporting the aforementioned studies suggesting that these cells are the

AML LSCs They also demonstrated the clonal advantage of preleukemic

HSCs over non-mutated hematopoietic cells (NPM1 mutation allele

frequency in HSCs ~20 normal HSC allele frequency ~5) at

diagnosis and their expansion during relapse (NPM1 mutation allele

frequency in HSCs ~100) suggesting that the therapeutic regimens

currently used to treat AML select for the outgrowth of the preleukemic

HSCs (Shlush et al 2014 Kim et al 2000)

The aforementioned studies have yielded a conceptual framework for the

hierarchical organization in AML (Figure 4) AML is primarily a disease

of the elderly and is associated with the development of age-related

mutations (Caligiuri et al 1994) The vast majority of these molecular

aberrations are founder mutations that have no clinical implications Some

combinations of mutations however give rise to preleukemic HSCs that

develop a clonal advantage and give rise to expanded progenitors and

mature cells harboring these mutations Such expansion increases the

36

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

likelihood that additional mutations will accumulate in these preleukemic

populations (eg LMPPs and GMPs) and eventually lead to the

acquisition of a correct combination of class I class II class III andor

non-genetic lesions that are required for transformation into AML This

model of AML pathogenesis suggests that transformation occurs as a

series of events that are selected for via clonal evolution It also provides

an explanation for the molecular and phenotypic heterogeneity of LSCs

and leukemic blasts that likely contribute to the patient-to-patient

variability in therapeutic responses

5 AML - Therapeutic Implications of the Cancer Stem Cell Theory

37

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

51 LSC Resistance to Conventional Therapies

50-80 of patients with AML experience CR with induction therapy

however most relapse within 3-5 years of their diagnosis Conventional

chemotherapies (eg cytarabine and anthracyclines) effectively reduce the

number of leukemic cells and thus form the basis for partial and complete

remission However these chemotherapies non-specifically target rapidly

proliferating cells As a consequence they fail to eradicate quiescent LSCs

and preleukemic HSCs (Guan et al 2003) LSCs have also been shown to

express multidrug resistant (MDR) pumps which provide LSCs with the

ability to pump cytotoxic agents out of the cytoplasm and into the

extracellular environment The up-regulation of NF-ĸB an inflammatory

transcription factor has also been shown to facilitate cell survival in LSCs

(Guzman et al 2001 Frelin et al 2005) Therefore clinical relapse is

likely caused by the persistence and outgrowth of chemotherapy-resistant

LSCs during remission (Figure 5) In support of this theory researchers

have identified the persistence of mutated hematopoietic cells during

remission and their outgrowth during relapse (Slush et al 2014)

While chemotherapy is required to prolong survival it may also accelerate

the clonal evolution of disease This idea has been supported by various

studies demonstrating the transformation of mice expressing preleukemic

lesions upon treatment with alkylating agents (Castilla et al 1999

38

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Rhoades et al 2000 Higuchi et al 2002) It has also been evidenced by

the outgrowth of leukemic cells with novel mutations in patients following

therapy (Shlush et al 2014) While not yet demonstrated experimentally

the DNA damaging properties of chemotherapy likely produce numerous

genetic lesions in preleukemic GMPs CMPs andor LMPPs as well as

pre-existing LSCs Some of these molecular aberrations likely give rise to

novel LSCs that undergo positive selection and lead to the outgrowth of

highly aggressive leukemic blasts that are resistant to AML therapies

(Figure 6) This likely accounts for the difficulty of inducing remission in

patients once they relapse Therefore the most effective treatment strategy

would entail the replacement of DNA damaging agents with LSC- and

molecular-directed therapies

52 Therapeutic Targeting of CD99

Monoclonal antibodies (mAbs) are commonly used to therapeutically

target cell surface proteins (eg CD20 in B cell lymphomas [rituximab]

WT1 in AML) (Stathis et al 2012 Oka et al 2004) Such mAb-directed

therapies effectively mediate cell death by directly inducing apoptosis

stimulating antigen-dependent cellular cytotoxicity (ADCC) activating

complement or by being conjugated to cytotoxic drugs (Smith 2003

Sutherland et al 2013) Several well-known LSC markers currently under

investigation as targets of mAbs include CD47 CD123 CD33 and CD44

39

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

(Majeti et al 2009 Jin et al 2009 Hauswirth et al 2007 Jin et al 2006)

In this section I will describe the mAb-targeting of CD99 a novel AML

LSC marker discovered in our laboratory

CD99 is a 32kDa cell surface protein expressed on leukocytes and

endothelial cells It mediates leukocyte transendothelial migration during

inflammation stimulates apoptosis in CD4+CD8+ thymocytes during

negative selection and facilitates cell adhesion in peripheral T cells (Luo

et al 2007 Alberti et al 2002) It has been shown to block neural

differentiation in Ewingrsquos sarcoma and to serve as an effective target in

the treatment of B-cell acute lymphoblastic leukemia (B-ALL) (Rocchi et

al 2010 Husak et al 2010)

To identify a novel therapeutic target on AML LSCs we performed a

transcriptional analysis comparing patient AML LSCs to normal HSCs

and identified CD99 as an up-regulated target on both CD34+CD38- LSCs

and bulk leukemic blasts with the level of CD99 expression being

significantly higher on LSCs relative to the latter To functionally validate

CD99 as a marker of LSCs we performed a limiting dilution analysis that

revealed a 124401 LSC frequency among the highest fraction of CD99

expressers and a 0360000 LSC frequency in the lowest fraction using

NSG mice (Chung et al 2013) We also found that CD34+CD38- enriched

AML fractions expressing high CD99 resemble LMPPs (CD34+CD38-

40

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

CD90-CD45RA+) while low CD99 expressing cells enrich for normal

HSCs (Chung et al 2013) Therefore we phenotypically and functionally

validated the up-regulation of CD99 on LSCs

We have also demonstrated that CD99 is up-regulated on 81 of

diagnostic AML patient samples 83 of relapsed AML patient samples

and 1111 de novo human AML cell lines (Chung et al 2013) Moreover

the level of CD99 expression is greater on relapsed patient samples

relative to diagnostic samples suggesting that the targeting of CD99 may

be effective in both stages of disease To determine whether mAbs could

be used to effectively target CD99 in AML we screened both de novo

AML cell lines and CML cell lines in blast crisis with commercially

available CD99 mAbs To our surprise the mAbs mediated a significant

reduction in cell number in both AML cell lines with cells expressing

BCR-ABL (eg CML in blast crisis) demonstrating slight resistance

relative to de novo AML To validate the clinical relevance of CD99

targeting in AML we have shown that the CD99 mAb is significantly

cytotoxic to AML cell lines (eg HL60) and AML patient samples (n=7)

(Chung et al 2013) In addition CD99 mAb leads to the relatively sparing

of HSCs and HUVECs suggesting that there is a large therapeutic window

(Chung et al 2013) We are currently investigating the mechanism of

toxicity mediated by this drug

41

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

To evaluate whether CD99 has a prognostic value in AML we analyzed

the level of CD99 expression and correlated these findings with the

clinical outcomes of 358 patients enrolled in the Eastern Cooperative

Oncology Group (ECOG) (E1900) trial Our results indicate that the level

of CD99 expression directly correlates with clinical outcomes (Figure

11a) However treating low CD99 expressers with high-dose

daunorubicin (90mgm2 vs 45mgm2) during induction therapy improves

their prognosis (Figure 11b) In contrast CD99 high expressers do not

seem to benefit from high-dose daunorubicin (Chung et al 2013)

Collectively our preliminary data suggests that CD99 is a LSC marker

that is also expressed by leukemic blasts that the targeting of CD99 is

significantly cytotoxic to leukemic cells (cell lines and patient samples) in

vitro that there is a large therapeutic window and that the level of CD99

expression could be used to identify patients who would clinically benefit

from high-dose daunorubicin

6 Conclusions

42

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Insights into the pathogenesis of leukemia have evolved with the development of various

technologies The first karyotypic analyses identified AML as malignancies associated

with cytogenetic abnormalities These findings were followed by the identification of

recurrent somatic mutations using sequencing technologies Recently studies evaluating

global DNA methylation the BM microenvironment and microRNAs have revealed the

importance of epigenetic modifiers the BM environment and other non-genetic

alterations in driving leukemogenesis In addition advancements in the ability to isolate

distinct cell populations and to analyze them using in vitro and xenotransplantation

assays led to the discovery of LSCs and our current knowledge regarding the cellular

mechanisms underlying AML pathogenesis

Through these studies we have learned that AML is a complex disease that originates

from age-related genetic andor environmentally induced lesions in immature

hematopoietic cells that reside in the bone marrow Some of these lesions occur in HSCs

(also known as preleukemic HSCs) which gain a clonal advantage and give rise to

downstream committed progenitors harboring these mutations The clonal expansion of

the downstream progenitors increases the likelihood that additional mutations will

accumulate in these cells and the additional lsquohitsrsquo lead to the eventual development of

fully transformed leukemic stem cells which in turn give rise to leukemic blasts The

resulting accumulation of leukemic blasts in the bone marrow leads to life-threatening

cytopenias Chemotherapy is cytotoxic to leukemic blasts and thus reverses the

cytopenias however it fails to address the underlying cause of the malignancy In

addition chemotherapy seems to drive the clonal evolution of AML by generating and

43

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

selecting for novel LSC clones as well as leukemic blasts that are highly resistant to

therapy

To effectively eradicate AML patients must be accurately diagnosed based on the

cytogenetic epigenetic and molecular make-up of their malignancy and novel therapies

targeting LSCs and leukemic blasts must be administered using a personalized approach

The prognostication of patients based on their molecular profiles are continuing to

evolve Moreover a number of agents targeting leukemic cells are currently in the drug

development pipeline including small molecule inhibitors of DNMT3A FLT3-ITD

IDH12 and MLL fusions as well as antibodies targeting WT1 In addition anti-CD47

CD123 CD33 CD44 and CD99 antibodies are being investigated as potential LSC-

directed therapies While no single miracle pill will likely cure the disease (though

possible) we believe that personalized combinatorial strategies will significantly improve

the outcome of patients victimized by this life-altering disease For the first time in over

40 years the personalized targeting of LSCs and leukemic blasts is becoming a reality

Figures

44

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Figure 1 Incidence of AML in the US Children AYAs adults and older adults account for 36 42 97 and 826 of AML diagnoses in the US

Figure 2 HSCs undergo symmetric (a) and asymmetric (b) division during normal hematopoiesis Red arrows indicate self-renewal

45

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Figure 3 Normal hematopoiesis The hematopoietic system consists of rare quiescent HSCs that give rise to MPPs upon differentiation MPPs are capable of differentiating into LMPPs CMPs and CLPs LMPPs give rise to GMPs andor lymphocytes CLPs differentiate into lymphocytes and CMPs differentiate into GMPs andor MEPs GMPs give rise to the granulocytic and monocyte lineages while MEPs differentiate into cells of the erythrocytic and megakaryocytic lineages Red arrows indicate self-renewal

46

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Figure 4 Malignant hematopoiesis in AML (a) Normal hematopoiesis as described above (b) HSCs develop genetic lesions and thus become preleukemic HSCs (degHSC) Preleukemic HSCs differentiate into preleukemic MPPs (degMPP) and downstream progenitors (degGMP degLMPP) which harbor identical mutations to their original ancestor degHSC are clonally expanded and thus clonally expand their downstream progenitors This increases the possibility that the degGMPs will acquire additional mutations Some of these mutations lead to the acquisition of self-renewal and thus the development of LSCs (GMP-like LSC) (a) LSCs give rise to leukemic blasts that undergo maturation arrest and thus fail to differentiate into granulocytesmonocytes Alternatively the clonally expanded degLMPPs acquire additional mutations which provide the cells with the ability to self-renew (b) These LMPP-like LSCs give rise to GMP-like LSCs or degGMPs which give rise to leukemic blasts (b) Red arrows indicate self-renewal

47

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Figure 5 Proposed model for relapse following conventional chemotherapy At diagnosis the accumulation of malignant myeloblasts (leukemic blasts) in the bone marrow leads to cytopenias that lead to the signsymptoms of AML Chemotherapy eradicates bulk leukemic cells however LSCs persist during remission The LSCs eventually proliferate and lead to relapse Red arrows indicate self-renewal

Figure 6 Chemotherapy leads to the accumulation of mutations in pre-existing LSCs (a) and preleukemic progenitors (b) and thus produces a novel heterogeneous group of LSCs in the bone marrow Each of the downstream leukemic blasts harbor these novel mutations making it difficult to induce remission in patients following relapse Each color represents a novel mutation

48

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Tables

FAB Classificatio

n

Description

M0 AML Minimally DifferentiatedM1 AML without MaturationM2 AML with MaturationM3 Acute Promyelocytic LeukemiaM4 Acute Myelomonocytic LeukemiaM5 Acute Monoblastic and Monocytic

LeukemiaM6 Acute ErythroleukemiaM7 Acute Megakaryoblastic Leukemia

Table 1 FAB classification of AML subtypes (Arber et al 2003)

Risk Status Cytogenetics Molecular Abnormalities

Good-risk Inv(16) t(1616) t(821) t(1517)

Normal karyotype carrying NPM1 mutation or isolated

CEBPA mutation in the absence of FLT3-ITD

Intermediate-risk Normal karyotype +8 t(911) other non-defined

t(821) inv(16) or t(1616) with c-Kit mutation

Poor-risk Complex karyotype (ge3 clonal chromosomal

abnormalities) 5- 5q- 7- 7q- 11q23 ndash non t(911)

inv(3) t(33) t(69) t(922)

Normal karyotype carrying a FLT3-ITD mutation

Table 2 Current risk stratification of patients based on cytogenetic and molecular abnormalities (NCCN)

Cytogenetic Classification Mutations Overall Risk Profile

Favorable Any Favorable

FLT3-ITD-Mutant

NPM1 and IDH1 or

IDH2

Wild-type ASXL1

49

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Intermediate RiskNormal Karyotype

FLT3-ITD- MLL-PTD PHF6 and

TET2

Intermediate

FLT3-ITD +-

Mutant CEBPA

FLT3-ITD+

Wild-type MLL-PTD TET2 and DNMT3a

and trisomy 8-

FLT3-ITD-Mutant

TET2 MLL-PTD

ASXL1 or PHF6

FLT3-ITD+

Mutant TET2 MLL-

PTD DNMT3A or trisomy 8

without mutant CEBPA

Unfavorable Any UnfavorableTable 3 Risk stratification of intermediate-risk patients based on novel molecular aberrations (Patel et al 2012)

50

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

References

Adams F The Genuine Works of Hippocrates W Wood New York 1886

Alberti I Bernard G Rouquette-Jazdanian AK et al CD99 isoforms expression dictates T cell functional outcomes FASEB 2002 16 1946-48

Arber DA Stein AS Carter NH Ikle D Forman SJ et al Prognostic Impact of Acute Myeloid Leukemia Classification Am J Clin Pathol 2003119672-680

Becker AJ McCulloch EA Till JE Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells Nature19634866452-454

Beckerich F Sobh M Morisset S Plesa A Dubois V Mollet I et al A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytometry Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation Blood 2013122(21)4639

Bhatia M Bonnet D Murdoch B et al A newly discovered class of human hematopoietic cells with SCID-repopulating activity Nat Med 199841038-1045

Bitoun E Oliver PL Davies KE The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling Hum Molm Genet 20071692-106

Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nature Medicine 19973730-737

Breems DA Van Putten WL De Greef GE Van Zelderen-Bhola SL et al Monosomal karyotype in acute myeloid leukemia a better indicator of poor prognosis than a complex karyotype J Clin Oncol 200826(29)4791-7

Brown D Kogan S Lagasse E et al A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PNAS 1997 942551-2556

Buggins AGS Milojkovic D Arno MJ Lea NC et al Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-kB c-Myc and pRb Pathways J of Immun 2001167(10)6021-6030

Byrd JC Mrozek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B (CALGB 8461) Blood 20021004325ndash4336

51

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Caligiuri MA Schichman SA Strout MP et al Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations Cancer Res 1994 54370ndash373

Cashen AF Schiller GJ OrsquoDonnell MR DiPersio JF Multicenter phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia J Clin Oncol 201028556ndash561

Castilla LH Garrett L Adya N Orlic D et al The fusion gene CbfbndashMYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nature Genetics 199923144-146

Castilla LH Wijmenga C Wang Q Stacy T Speck NA Failure of embryonic hematopoiesis and lethal hemor- rhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Castilla LH Wijmenga C Wang Q Stacy T et al Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knock-in leukemia gene CBFbndashMYH11 Cell 199687687ndash696

Cheng J Guo S Chen S Mastriano SJ et al An Extensive Network of TET2-Targeting MicroRNArsquos Regulates Malignant Hematopoiesis Cell Reports 20135471-481

Christianson SW Greiner DL Hesselton RA et al Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice J Immunol 19971583578-3586

Chung S Tavakkoli M Devlin SM Park CY CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes In 55th

ASH Annual Meeting and Exposition Dec 8 2013 Ernest N Morial Convention Center Blood Abstract 2891

Cole M Strair R Acute myelogenous leukemia and myelodysplasia secondary to breast cancer treatment case studies and literature review Am J Med Sci 2010339(1)36-40

Cozzio A Passegue E Ayton PM et al Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Genes Dev 200317(24) 3029-35

Dosil M Wang S Lemischka IR Mitogenic signaling and substrate specificity of the Flk2Flt3 receptor kinase in fibroblasts and interleukin 3-dependent hematopoietic cells Mol Cell Biol 199313(10)6572-85

Doulatov S Notta F Laurenti Dick JE et al Hematopoiesis A Human Perspective Cell Stem Cell 201210(2)120-136

52

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Duval M Klein JP He W Cahn JY et al Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure JCO 201028(23)3730-3738

Eppert K Takenaka K Lechman ER Waldron I Nillson B et al Stem cell gene expression programs influence clinical outcome in human leukemia Nature Medicine 2011 17(9) 1086-93

Erfurth F Hemenway CS de Erkenez AC Domer PH MLL fusion partners AF4 and AF9 interact at subnuclear foci Leukemia 20041892ndash102

Fernandez HF New Trends in the Standard of Care for Initial Therapy of Acute Myeloid Leukemia ASH Education Book 20102010(1)56-61

Fleming WH Alpern EJ Uchida N Ikuta K Spangrude GJ Weissman IL Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells J Cell Biol 1993122(4)897-902

Frelin C Imbert V Griessinger E Peyron AC Rochet N et al Targeting NF-ĸB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood 2005105(2)804-811

Garzon R Liu S Fabbri M Liu Z Heaphy CE et al MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1 Blood 2009b113(25)6411-8

Gilliland DG Hematologic malignancies Current Opinions in Haematology 20018189-191

Goardon N Marchi E Atzberger A Quek L et al Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia Cancer Cell 2011 19 138-152

Greenblatt S Li L Slape C Nguyen B et al Knock-in of a FLT3ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model Blood 20121192883-2894

Greenblatt SM Nimer SD Chromatin modifiers and the promise of epigenetic therapy in acute leukemia Leukemia 20141-11

Grimwade D Walker H Oliver F et al The importance of diagnostic cytogenetics on outcome in AML analysis of 1612 patients entered into the MRC AML 10 trial The Medical Research Council Adult and Childrenrsquos Leukaemia Working Parties Blood 1998922322ndash2333

Grimwade D Walker H Harrison G Oliver F et al The predictive value of hierarchical

53

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

cytogenetic classification in older adults with acute myeloid leukemia (AML) analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial Blood 200198(5)1312-1302

Gu X Mahfouz RZ Enane F Hu Z et al A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation Blood 2013122(21)1254

Guan Y Gerhard B Hogge DE Detection isolation and stimulation of quiescent primitive leu- kemic progenitor cells from patients with acute myeloid leukemia (AML) Blood 1013142-3149 2003

Gurchot C The trophoblastic theory of cancer (John Beard 1857-1924) revisited Oncology 197531(5-6)310-33

Guzman ML Neering SJ Upchurch D Grimes B Howard DS et al Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells Blood 200115(98)2301-7

Hagemann S Heil O Lyko F Brueckner B Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines PLoS One 20116(3)e17388

Hajdu SI The First Tumor Pathologist Association of Clinical Scientists 2004 34(3)355-356

Han YC Park CY Bhagat G Zhang J Wang Y Fan JB et al microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors biased myeloid development and acute myeloid leukemia J Exp Med 2010207(3)475-489

Hăulică I Petrescu G The kidneymdashan endocrine organ Rev Med Chir Soc Med Nat Iasi 198589(2)213-21

Hauswirth AW Florian S Printz D Sotlar K Krauth MT et al Expression of the target receptor CD33 in CD34+CD38-CD123+ AML stem cells Eur J Clin Invest 200737(1)73-82

Higuchi M OrsquoBrien D Kumaravelu P Lenny N et al Expression of a conditional AML1ndashETO oncogene by-passes embryonic lethality and establishes a murine model of t(821) acute myeloid leukaemia Cancer Cells 2002163ndash74

Hirade T Abe M Onishi C Yamaguchi S et al Flt3ITD Blocks Myeloid Differentiation Of Hematopoieitic Cells By Up-regulating Runx1 In 55th ASH Annual Meeting and Exposition Dec 9 2013 Ernest N Morial Convention Center Blood Abstract 3803

Howlader N Noone AM Krapcho M Garshell J Neyman N Altekruse SF Kosary CL

54

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Yu M Ruhl J Tatalovich Z Cho H Mariotto A Lewis DR Chen HS Feuer EJ Cronin KA (eds) SEER Cancer Statistics Review 1975-2010 National Cancer Institute Bethesda MD httpseercancergovcsr1975_2010 based on November 2012 SEER data submission posted to the SEER web site April 2013

Huntly BJP Gilliland DG Leukaemia stem cells and the evolution of cancer-stem-cell research Nat Rev Cancer 2005 5(4)311-21

Husak Z Printz D Schumich A Potschger U Dworzak MN Death induction by CD99 ligation in TELAML1-positive acute lymphoblastic leukemia and normal B cell precursors J Leukoc Biol 2010 88(2)405-12

Inoue K Tamaki H Ogawa H Oka Y Soma T et al Wilmsrsquo tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells Blood 199891(8)2969-76

Ishii M Matsuoka Y Sasaki Y Nakatsuka R et al Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells Experimental Hematology 201139(2)203-213

Ishikawa F Yoshida S Saito Y Hijikata A Kitamura H Tanaka S et al Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region Nature Biotechnology 2007251315-1321

Itzykson R Kosmider O Cluzeau T et al Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias Leukemia 2011 251147ndash1152

Jan M Snyder TM Corces-Zimmerman MR Vyas P et al Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia Leukemia 20124(149)149ra118

Jawad M Giotopoulos G Cole C Plumb M Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis Br J Radiol 200780(1)S56-62

Jin L Lee EM Ramshaw HS Busfield SJ et al Monoclonal Antibody-Mediated Targeting of CD123 IL-3 Receptor α Chain Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 20095(1)31-42

Jin L Hope KJ Zhai Q Smadja-Joffe F Dick JE Targeting of CD44 eradicates human acute myeloid leukemia stem cells Nature Medicine 2006121167-1174

Josting A Wiedenmann S Franklin J et al Secondary myeloid leukemia and myelodysplastic syndromes in patients treated for Hodgkinrsquos disease a report from the German Hodgkinrsquos Lymphoma Study Group J Clin Oncol 200321(18)3440-6

55

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Kats LM Reschke M Taulli R Pozdnyakova O et al Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance Cell Stem Cell 201414(3)329-41

Kayser S Zucknick M Dohner K Krauter J et al Monosomal karyotype in adult acute myeloid leukemia prognostic impact and outcome after different treatment strategies Blood 2012119(2)551-8

Kelly LM Kutok JL Williams IR Boulton CL Amaral SM et al PMLRARa and FLT3-ITD induce an APL-like disease in a mouse model PNAS 2002a998283ndash8288

Kelly LM Liu Q Kutok JL Williams IR et al FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myelopro- liferative disorders in a murine bone marrow transplant model Blood 2002b99310ndash318

Kern W Haferlach T Schnittger S Hiddemann W Schoch C Prognosis in therapy-related acute myeloid leukemia and impact of karyotype J Clin Oncol 200422(12)2510-11

Khalade A Jaakkola MS Pukkala E Jaakkola JJ Exposure to benzene at work and the risk of leukemia a systematic review and meta-analysis Environ Health 2010931

Kim HJ Tisdale JF Wu T Takatoku M et al Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates Blood 2000961ndash8

Klusmann JH Li Z Bohmer K Maroz A Koch ML Emmirch S et al miR-125b-2 is a potential oncomiR on human chromsome 21 in megakaryoblastic leukemia Genes Dev 201024(5)478-90

Ko M Huang Y Jankowska AM Pape UJ Tahiliani M et al Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 Nature 2010468839ndash843

Ko M Bandukwala HS An J Lamperti ED Thompson EC Hastie R et al Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice PNAS 201110814566ndash14571

Kode A Manavalan JS Mosialou I Bhagat G et al Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts Nature 2014506240-4

Kogan SC Brown DE Schultz DB et al BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor 1113098 chimeric protein (PMLRARα) to block neutrophilic differentiation and initiate acute leukemia J Exp Med 2001193531-543

Kogan SC Lagasse E Atwater S Bae SC Weissman I et al The PEBP2bndashMYH11 fusion created by inv(16)(p13q22) in myeloid leukemia impairs neutrophil matur- ation

56

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

and contributes to granulocytic dysplasia PNAS 19989511863-11868

Koszarska M Bors A Feczko A Meggyesi N Batai A Csomor J et al Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia Leukemia amp lymphoma 201354(5)1028-1035

Kottaridis PD Gale RE Frew ME Harrison G Langabeer SE et al The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy analysis of 854 patients from the United Kingdom Research Council AML 10 and 12 trials Blood 2001981752-1759

Krause DS Ito T Fackler MJ Smith OM Collector MI Sharkis SJ May WS Characterization of murine CD34 a marker for hematopoieitic progenitor and stem cells Blood 1994 84(3)691-701

Kreipe HH Precursors of acute leukemia myelodysplastic syndromes and myeloproliferative neoplasms Pathologe 201132271-6

Krivtsov AV Armstrong SA MLL translocations histone modifications and leukaemia stem-cell development Nature Reviews 20077(11)823-833

Krivtsov AV Twomey D Feng Z Stubbs MC et al Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9 Nature 2006 442(7104) 818-22

Lapidot T Sirard C Vormoor J Murdoch B et al A cell initiating human acute myeloid leukemia after transplantation in SCID mice Nature 1994367645-648

Lee BH Williams IR Anastasiadou E et al FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model Oncogene 200524(53)7882-92

Ley TJ Ding L Walter MJ McLellan MD Lamprecht T et al DNMT3A mutations in acute myeloid leukemia NEJM 20103632424-2433

Levis M FLT3 mutations in acute myeloid leukemia what is the best approach in 2013 ASH Education Book 20132013(1)220-226

Litzow MR Tarima S Perez WS Bolwell BJ Cairo MS et al Allogeneic transplantation for therapy-related myelodysplastic syndorem and acute myeloid leukemia Blood 2010 115(9)1850-1857

Luo O Alcaide P Luscinskas FW Muller WA CD99 is a key mediator of the transendothelial migration of neutrophils J Immunol 2007178(2)1136-43

57

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Mailander V Scheibenbogen C Thiel E et al Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia 200418(1)165-6

Majeti R Park CY Weissman IL Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood Cell Stem Cell 20071635ndash645

Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S et al CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells Cell 2009138(2)286-99

Marchetto S Fournier E Beslu N et al SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor Leukemia 199913(9)1374-82

Martelli MP Pettirossi V Thiede C Bonifacio E Mezzasoma F et al CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice Blood 20101163907ndash3922

Matsuno N Osato M Yamashita N Yanagida M Nanri T et al Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype Leukemia 2003172492ndash2499

Medeiros BC Othus M Fang M et al Prognostic impact of monosomal karyotype in young adult and elderly acute myeloid leukemia the Southwest Oncology Group (SWOG) experience Blood 20101162224-8

Metzeler KH Walker A Geyer S et al DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia Leukemia 2012261106-1107

Miraki-Moud F Anjos-Afonso F Hodby KA et al Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation PNAS 2013110(33)13576-81

Miyamoto M Weissman IL Akashi K AMLETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 821 chromosomal translocation PNAS 200097(13)7521-7526

Moran-Crusio K Reavie L Shih A Abdel-Wahab O Ndiaye-Lobry D Lobry C et al Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation Cancer Cell 20112011ndash24

Mousquet M Quelen C Rosati R Mansat-De Mas V et al Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(211)(p21q23) translocation J Exp Med 2008205(11)2499-506

58

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

National Comprehensive Cancer Network Acute Myeloid Leukemia (Version 22014) httpwwwnccnorgprofessionalsphysician_glsPDFamlpdf Accessed May 12 2014

Nguyen AT Taranova O He J Zhang Y DOT1L the H3K79 methyltransferase is required for MLL-AF9-mediated leukemogenesis Blood 2011117(25)6912-22

Notta F Doulatov S Laurenti E Poeppi A Jurisica I Dick JE Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 2011333218-221

Office for National Statistics (2004) Cancer Statistics Registrations Registrations of Cancer Diagnosed in 2003 England National Statistics Series MB1 London 34

Oka Y Tsuboi A Taguchi T Osaki T et al Induction of WT1 (Wilmsrsquo tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression 2004101(38)13885-90

Okada S Nakauchi H Magayoshi K Nishikawa S Miura Y Suda T In Vivo and In Vitro Stem Cell Function of c-kit ndash and Sca-1 Positive Murine Hematopoietic Cells Blood 199280(12)3044-3050

Okada Y Feng Q Lin Y Jiang Q Li Y Coffield VM et al hDOT1L links histone methylation to leukemogenesis Cell 2005121167ndash178

Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by single CD34-lownegative hematopoietic stem cell Science 1996273 242-245

Owen CJ Toze CL Koochin A Forrest DL Smith CA et al Five new edigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy Blood 2008 1124639-4645

Pabst T Eyholzer M Fos J Mueller BU Heterogeneity within AML with CEBPA mutations only CEBPA double mutations but not single CEBPA mutations are associated with favourable prognosis BJC 20091001343-1346

Patel JP Goumlnen M Figueroa ME Fernandez H et al Prognostic relevance of integrated genetic profiling in acute myeloid leukemia NEJM 2012366(12)1079-89

Plzak LF Erythropoietin-a renal hormone Surg Forum 196010121-4

Pronier E Almire C Mokrani H Vasanthakumar A Simon A da Costa Reis Monte Mor B et al Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors Blood 20111182551ndash2555

59

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Qin F Shao H Chen X Tan S et al Knockdown of NPM1 by RNA Interference Inhibits Cells Proliferation and Induces Apoptosis in Leukemic Cell Lines Int J Med Sci 20118(4)287-294

Ravandi F Patel K Luthra R Faderl S Konopleva M Kadia T et al Prognostic significance of alterations in IDH enzyme isoforms in patients with AML treated with high-dose cytarabine and idarubicin Cancer 2012118(10)2665-2673

Reilly JT Pathogenesis of acute myeloid leukemia and inv(16)(p13q22) a paradigm for understanding leukemogenesis BJH 2004 12818-34

Rhoades KL Hetherington CJ Harakawa N Yergeau DA et al Analysis of the role of AMLndashETO in leukemogenesis using an inducible transgenic mouse model Blood 2000962108ndash2115

Rocchi A Manara MC Sciandra M Zambelli D et al CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis J Clin Invest 2010120(3)668-680

Rucker FG Schlenk RF Bullinger L Kayser S Teleanu V et al TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations Monosomal Karyotype and Dismal Outcome Blood 2012119(9)2214-21

Sasaki M Knobbe CB Munger JC Lind EF et al IDH1(R132H) mutation increases murine hematopoietic progenitors and alters epigenetics Nature 2012488(7413)656-9

Scheijen B Ngo HT Kang H Griffin JD FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins Oncogene 2004233338-3349

Selig C Nothdurft W et al Cytokines and progenitor cells of grnulocytopoiesis in peripheral blood of patients with bacterial infections Infect Immun 199563(1)104-109

Shih AH Abdel-Wahab O Patel JP Levin RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 201212(9)599-612

Shin YS Hu W Naramura M Park Y High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias 2014 211(2) 217-231

Shlush LI Zandi S Mitchell A Chen WC Brandwein JM et al Identification of pre-leukaemic haematopoietic stem cells in acute leukemia Nature 2014506328-333Shultz DB Phan VT Truong B-TH Kogan SC Cytokine stimulation cooperates with PMLRARα to cause leukemia Blood 200096573a

60

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Shultz LD Lyons BL Burzenski LM Gott B Chen X et al Human lymphoid and myeloid cell development in NODLtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells J Immunol 2005174(10)6477-89

Sieburg HB Cho RH Muller-Sieburg CE Limiting dilution analysis for estimating the frequency of hematopoietic stem cells uncertainty and significance Exp Hematol 200230(12)1436-43

Slovak ML Kopecky KJ Cassileth PA et al Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia a Southwest Oncology GroupEastern Cooperative Oncology Group Study Blood 2000964075-4083

Smith MR Rituximab (monoclonal anti-CD20 antibody) mechanism of action and resistance Oncogene 2003227359-7368

Solary E Bernard OA Tefferi A Fuks F Vainchenker W The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases Leukemia 201428485-496

Southam CM Brunschwig A Quantitative Studies of Autotransplantation of Human Cancer Preliminary report Cancer 196114971-978

Stacchini A Fubini L Severino A Sanavio F et al Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts Leukemia 1996101584-1591

Stathis A Ghielmini New agents for the treatment of lymphoma Annals of Oncology 201223(suppl 10)92-98

American Association for Cancer Research (AACR) (2014) First-in-class Cancer Metabolism Drug AG-221 Shows Clinical Activity in Advanced Blood Cancers Retrieved from httpwwwaacrorghomepublic--mediaaacr-in-the-newsaspxd=3308

Sutherland MSK Walter RB Jeffrey SC Burke PJ Yu C Kostner H Stone I et al SGN-CD33A a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML Blood 2013122(8)1455-1463

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 200096(4)1254-1258

Tallman MS Bowlings PA Milone G Zhang MJ et al Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission Blood 2000 96(4)1254-1258

61

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Taskesen E Bullinger L Corbacioglu A Sanders MA Erpelinck CAJ et al Prognostic impact concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients further evidence for CEBPA double mutant AML as a distinctive disease entity Blood 2011117(8)2469-2475

Taussig DC Miraki-Moud F Anjos-Afonso F et al Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells Blood 2008112(3)568ndash575

Taussig DC Vargaftig J Miraki-Moud F Griessinger E Sharrock K et al Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34- fraction Blood 2009115(10)1976-1984

Thol F Damm F Ludeking A Winschel C et al Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia J Clin Oncol 201129(21)2889-96

Thompson CB Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia Blood 2013122(21)SCI-26

Tsuboi A Oka Y Katayama Y Elisseeva OA et al Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease Leukemia 2012261410-1413

Wang JCY Dick JE Cancer stem cells lessons from leukemia Trends Cell Biology 200515(9)494-501

Wang X Gong J Yu J Wang F Zhang X et al MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia Blood 20121194992-5004

Yagasaki H Mugishima H Hereditary diseases with propensity to myeloid malignancy Nihon Rinsho 200967(10)1884-8

Yahata T Ando K Sato T Miyatake H Nakamura Y et al A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NODSCID mice bone marrow Blood 2003101(8)2905-2913

Yamauchi T Negoro E Lee S Takai M Matsuda Y et al Detectable Wilmrsquos tumor-1 transcription at treatment completion is associated with poor prognosis of acute myeloid leukemia a single institutionrsquos experience Anticancer Res 201333(8)3335-40

Zanjani ED Almeida-Porada G Livingston AG Flake AW Ogawa M Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells Exp Hematol 1998 26(4) 353-360

62

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63

Zeisig DT Bittner CB Zeisig BB Garcia-Cuellar MP et al The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin Oncogene 200524 5525ndash5532

Zhang W Konopleva M Shi YX McQueen T et al Mutant FLT3 a direct target of sorafenib in acute myelogenous leukemia J Natl Cancer Inst 2008100(3)184-198

Zhang W Xia X Reisenauer M et al Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner J Biol Chem 200628118059-18068

Ziaodong L Xin Y Mi R Ding J Wang X et al Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia PLOS one 20149(3)e92470

63