Pathogenetic Features of Hematopoiesis in MDS: Focus …€¦ · Pathogenetic Features of...

Pathogenetic Features of Hematopoiesis in MDS: Focus on Aging

Irving L. Weissman, MDStanford University School of Medicine

Director, Institute for Stem Cell Biology

and Regenerative Medicine

Director, Stanford Ludwig Center for Cancer

Stem Cell Research and Medicine

Professor of Pathology and Developmental Biology

Stanford, California

DISCLOSURE

I have no relevant financial relationships to disclose.

Blood-Forming Stem Cells

Ludwig Center at Stanford University

IN VIVO

VERITAS

1. Development

2. Maintenance

3. Malignant

transformation

Blood forming stem cells make blood, and only blood

HSC

MultipotentProgenitors

CLP

T-cells NK-cellsB-cells

MEP

GMP

CMP

OligopotentProgenitors

Mature effector cells

Pro-T Pro-NKPro-B

Dendriticcells

Pro-DC

Lineage restrictedProgenitors

GranulocytesErythrocytesPlatelets Macrophages

Hematopoietic Hierarchyhttp://gexc.stanford.edu

Lin-c-Kit+Sca1- IL7Ra- CD34- FcgR+

Lin-c-Kit+Sca1- IL7Ra- CD34-FcgR-Slamf1+

Lin-c-Kit+Sca1-/lo IL7Ra- CD34+ FcgRlowSlamf1-Tie2+

Lin-c-Kit+Sca1+ IL7Ra- CD34+Slamf1+Tie2+

Lin-c-Kit+Sca1+IL7Ra-CD34-Slamf1+Tie2+

Lin-c-Kit+Sca1+ IL7Ra- CD34+Slamf1-Tie2-

Lin-c-KitloSca1loIL7Ra+Flk2hi

Lin-c-Kit+Sca1+ IL7Ra- CD34+Slamf1-Tie2+

Lin-CD34+CD38-CD90+CD45RA-Mouse

Human

Lin-CD34+CD38-CD90-CD45RA-

Lin-CD34+CD38+CD10+

Lin-CD34+CD38+IL3RaCD45RA-

Lin-CD34+CD38+IL3RaCD45RA+

Lin-CD34+CD38+IL3RalowCD45RA-

EPMkP

Long-termSelf-renewal

GP MacP

PreGMPPreMEP

Lin-c-Kit+Sca1-/lo IL7Ra- CD34+ FcgRlowSlamf1+Tie2+

Lin-c-Kit+Sca1-/lo IL7Ra- CD34+ FcgRlowSlamf1-Tie2-

All isolated

Akashi, Kondo,

Traver, Seita,

Miyamoto, Bryder,

Spangrude, Ikuta,

Morrison, et al

A Model of HSC Ontogeny

Young adult Mid-age Old age

Lymphoid gene expression

Myeloid gene expression

Leukemia genes

Self-renewal

Myeloid potential

Myeloid genes

Leukemia genes

Lymphoid potential

Homing

Lymphoid genes

Rossi, Bryder,

Weissman,Seita, Beerman

Cell intrinsic changes

)

1) The cell-intrinsic functional and molecular properties of aging LT-HSC places the cellular and molecular rationale for the decreased immuno-competence that accompanies aging at the level of the LT-HSC.

2) Age-dependent upregulation of genes frequently activated as proto-

oncogenes in leukemogenesis (17/32 highest.)

3) ..and quiescent aged HSC build up DNA DS breaks, repaired upon G0

to G1 to S transition

4) Is this maturation of all HSC, or clonal selection of some HSC?

Clonal Selection or epigenetic programming of

HSC with Aging

Normal HSC 2 mo Normal HSC 2 years

Hypothesis and data: Rossi, Eaves, Muller-Sieburg, Nakauchi, Bryder,

Beerman, and Weissman

Leukemic cells in AML patients

CD34+CD38-Thy+Lin-CD34+CD38-Thy-Lin-

5-40%

AML-1/ETO+

Normal colonies Leukemic blast colonies

Miyamoto, Weissman

Akashi, PNAS 2000: 97: 6924

LT STEM CELLS MPP

Leukemia Stem Cells [LSC]

Cell of origin – progression to leukemia

Jak2

W

e

i

CML• Fialkow: clonal disorder in G,M.E,B cells; Rowley/Nowell

bcr-abl translocation; fusion protein in chronic, myeloproliferative phase; LSC proposed to be HSC or MPP; Jamieson and Weissman HSC.

• No increase in CD34+38lo90+Lin- frequency: competitive advantage vs nl HSC

• Myeloid blast crisis is at the stage of GMP, and overexpress activated β-catenin;axin inhibits them.

• 4/7 pts overexpress β-catenin by mis-splicing GSK3 the other inhibitor of β-catenin

Jamieson and Weissman, 2004, 2009

GSK30 10

210

310

410

5

<Alexa 405-A>: GSK3b

0

20

40

60

80

100

% o

f M

ax

CP CML

AP CML

BP CML

0 102

103

104

105

<FITC-A>: CD45RA

0

102

103

104

105

:

CD

12

3

5.98 90.3

0 102

103

104

105

<FITC-A>: CD45RA

0

102

103

104

105

:

CD

12

3

29.6

35.4 27.5

CD45RA

CD

123

GMPCMP

MEP

CMP GMP

MEP

A. Progenitor FACS Analysis B. Q-PCR of GSK3 Expression C. GSK3 FACS

Normal Progenitors CML BC Progenitors

D. Confocal Analysis of -catenin Expression

Normal GMP CML GMP

E. GSK3 cDNA Sequencing Analysis

Figure 3. Aberrant GSK3Expression by Blast Crisis CML Progenitors

AML Gene Annotation

SU070 PXDN V616I

KALRN S44P

TET2 Y1649stop

TET2 T1884A

TMEM8B nt G471A

NCRNA00200 nt G354A

TMEM20 A143T

ZRANB1 nt G4659A

SCN4B H227N

GABARAPL1 nt C1583T

DOCK9 A1475V PLAG2G4D P246A

CACNA1H R1069stop

CTCF R339Q GZF1 nt G3835C

PRPF6 R527H

CXorf36 I225L CXorf66 G321S

FLT3 599-610 ITD !

AML HSC

FACS

Normal

Tissue

(T Cells)

Next generation

exome sequencing

N Mutations

In AML

Identification of Somatic Mutations by Exome

Sequencing

Max Jan

Thomas Snyder

Ryan Corces-

Zimmerman

Steve Quake

Ravi Majeti

Irv Weissman Science Translatinal Medicine

2012 4(149): 149ra18.

Analysis of Single HSC to Identify Pre-Leukemic Clones

Max Jan

Thomas Snyder

Ryan Corces-

Zimmerman

Steve Quake

Ravi Majeti

TET2

E1357stop

TET2

D1384V,

SMC1A

FLT3,

NPM1

Leukemic

Clonal

Expansion

SKP2 ELP2

PDZD3

FLT3 Leukemic

Clonal

Expansion

Normal HSC----------- Stages of preleukemia progression in HSC clone--- LSC at progenitor stage

Ryan corces –Zimmerman, Ravi Majeti, and IW

Now 21 AMLs PNAS 2014

If you want to know which genes are likely oncogenes, ask the cancer.

1 In AML, progression is in a blood stem cell clone, while the leukemia stem cells is at the progenitor stage: preleukemic clones of HSC

compete with nl HSC

2 There are no leukemias we have foundthat are leukemias of HSC

3 If this is true for leukemia, it isprobably true for all cancers in tissuesthat regenerate from tissue stem cells

Lessons for leukemia and perhaps all cancers

CD47 was discovered as a marker of aging RBC by Oldenborg. We found it on m/h AML LSC

Macrophage

SIRPa

Receptor+

AMLLSC

CD47

Stimulus

---

Net Result: No Phagocytosis

Hypothesis: Increased expression of CD47 on myeloid leukemia cells contributes to pathogenesis by facilitating evasion of phagocytosis

Prediction: Increased expression of CD47 on human AML is associated with a worse clinical outcome

Eat me

Don’teat me

Programmed cell removal

Traver and IW 1998; Jaiswal, Majeti, Chao,and IW 2008.

TET47 MOLM 24h

TET47 MOLM 24h

200X

400X

TET MOLM 24h

TET MOLM 24h

200X

400XChao, Jaiswal, Majeti, Weissman

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R-femur R-tibia R-humerus L-femur L-tibia LInjection site

TetCD4

7

MOLM

Tet

MOLM

huCD45

Jaiswal et al 2009 Cell. 138(2): 271-85. l

Anti-CD47 Antibodies Enable Phagocytosis of AML LSC

Human Macrophages

NBM1

NBM2

NBM3

NBM4

SU001

SU008

SU009

SU014

SU016

SU018

SU028

SU032

SU035

X

Mark Chao, Majeti et al

Anti-CD47 Antibody Depletes AML in the Bone Marrow

IgG Control Anti-CD47 Anti-CD47

Mark Chao, Majeti et al Cell 2009 Cell. 138(2): 286-99

Inject Tumor Cells

Into Peritoneal Cavity

Initiate Treatment Evaluate Tumor Growth

With Bioluminescence

Imaging

2.5 Months2 Weeks

Volkmer, Willingham, Chin, and IW

Inject Tumor Cells Into

Left Hemisphere

Initiate Treatment Evaluate Tumor

Formation

8 Weeks2 Weeks

Monitor Tumor Growth

With Bioluminescence

imaging

MITRA, JIANG, CHESHIER, RAVEH, IW

Investigation and Targeting of CD47 in Human Cancers

GlioblastomaMedulloblastomaOligodendrogliomaHepatocellular CarcinomaGastric CancerMultiple MyelomaChronic Myeloid LeukemiaAcute Myeloid LeukemiaNon-Hodgkin’s Lymphoma

T-Acute Lymphoblastic LeukemiaB-Acute Lymphoblastic Leukemia

In all cases tested with metastatic tumors, the metastases areeliminated.

Breast OvarianBladderPancreaticColonProstateLungKidneyLeiomyosarcomaHead & NeckMelanoma

Monotherapy trials

Precancer cells express calreticulin, and emergent

cancer clones overcome this with CD47

lrl

Macrophage

SIRPa

Receptor+

AMLLSC

CD47

Stimulus

---

Increased expression of CD47 on myeloid leukemia cells contributes to

pathogenesis by facilitating evasion of phagocytosis

Eat me

Don’t

eat me

Lrp 1 calreticulin

Chao, Jaiswal, Weissman-Tsukamoto, Majeti, and IW

Science Transl Med 2010. 2(63)

Toll like receptor agonist incubation of macrophages increases anti-CD47

mediated phagocytosisof cancer cells

Mingye Feng, Volkmer, Weissman

TLR agonists synergize with anti-CD47to eliminate a prostate cancer

xenograft in vivo

BTK is expressed in many blood lineage cells, including macrophages

Phagocytosis of tumor cells is inhibited by the BTK inhibitor, ibrutinib


Anti calreticulin blocking antibody inhibits phagocytosis when incubated

with macrophages, but not with tumor cells


Precancer cells express calreticulin, and emergent

cancer clones overcome this with CD47

lrl

Macrophage

SIRPa

Receptor+

AMLLSC

CD47

Stimulus

---

Increased expression of CD47 on myeloid leukemia cells contributes to

pathogenesis by facilitating evasion of phagocytosis

Eat me

Don’t

eat me

Lrp 1 calreticulin

Calreticulin is an ER protein that is secreted following TLR to BTK

signalling; BTK phosphorylates calreticulin, which in the ER is

leaved to separate from KDEL domain. pY-calR binds to lrp-1 or

tumor cell. Mingye Feng,J.Volkmer, and Weissman: PNAS 2015

0%

10%

20%

30%

40%

Normal age-matched MDS

HS

C /

CD

34+

F

requency

A HSC frequency is similar in MDS and age-matched controls

N.S.

B MDS clone dominates HSC pool

0%

1%

10%

100%

NBM 7(-) MDS

hC

D45 C

him

erism

/ 5

00

HS

C

N.S.

0 20 40 60 80 100

SU03

SU02

SU01

% of total

7(-) nuclei Normal nuclei

C Xenotransplantation chimerism D Clonal dominance post-xenotransplantation

0 20 40 60 80 100

SU03-NSG2

SU03-NSG1

SU02-NSG2

SU02-NSG1

SU01-NSG2

SU01-NSG1

% of total

7(-) nuclei normal nuclei

Wendy Pang, John Pluvinage, Chris Park, IW, et al

MDS is a pre-AML disease of older patients in which a cytopeniaPrecedes leukemia.

MDS HSC outcompete normal HSC in MDS patients and in transplanted NSG mice

NBM 34+38+ MDS 34+38+

0

10

20

30

40

50

60

Normal HSC MDS HSC

Rela

tive M

FI

0

10

20

30

40

50

60

70

Normal 34+38+ MDS 34+38+

Rela

tive M

FI

0

20

40

60

80

100

Normal GMP MDS GMP

Rela

tive M

FI

0

20

40

60

80

100

Normal 34+38+ MDS 34+38+

Phagocytic I

nex

0

10

20

30

40

Normal 34+38+ MDS 34+38+

Phagocytic I

nex

E CRT-blocking peptide abrogates phagocytosis of MDS CD34+CD38+ cells

D Increased phagocytosis of MDS CD34+CD38+ cells C Increased CRT on MDS GMP

A CRT not increased on MDS HSC B Increased cell-surface CRT on MDS CD34+CD38+ cells

N.S. *

** ***

****

% o

f

ma

xim

um

CRT Expression

Normal + PBS

MDS + PBS

Normal + CRT-blocking

MDS + CRT-blocking

Wendy Pang, John Pluvinage,Chris Park, IW, et al

Calreticulin expressed onMDS progenitors, but notHSC, causes programmedcell removal

Wendy Pang, Weissman, Schrier

Conclusions

MDS HSC outcompete normal HSC in patient and xenotransplants

The MDS-initiating cell resides in the HSC compartment

High CRT predisposes MDS myeloid [GMP, MkP, EP] progenitors for programmed cell removal

Increased CD47 expression is a crucial step in the progression from MDS to AML

• Calreticulin and LRP1 are potential therapeutic targets

Wendy Pang, John Pluvinage, Chris Park, IW, et al

BCL2 blocks apoptosis, but not programmed cell removal

of neutrophiles: Lagasse and Weissman JEM 1994

Apoptotic alive

PCDeath and PCRemoval

• PCD is accompanied by PCR; blocking PCD with bcl2 does NOT block PCR [Lagasse and Weissman 1994, JEM]. PCR prevents inflammation.

• All cancers defeat PCD: p53, bcl2, bax,NFkB, etc

• All cancers defeat PCR: calreticulin, Ph-serine, asialoglycoprotein are ‘eat me’ signals countered by the CD47 ‘don’t eat me’ signal.

• Stimuli that induce PCD and/or PCR develop cell competition/selection in pre-cancer lineages that can result in cancer clones

Lokey Stem Cell Institute at Stanford

Ludwig Center at Stanford

Institute for Stem Cell Biology and Regenerative Medicine

Supported grants from NCI, NHLBI, Calif Inst Reg Medicine, Lacob Foundation, Siebel SCI

Wendy Pang, Weissman, Schrier

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