The bone marrow microenvironment in myeloid malignancies€¦ · 12 c a p i l l a r i e s (n o r m...

HSC

Progenitors

MSC

Lectures in Cancer Biology and Medicine

Sunday 2nd April 2020

The bone marrow microenvironment in myeloid malignancies

Simón Méndez-Ferrer, PhD

Department of Haematology

WT-MRC Cambridge Stem Cell Institute

University of Cambridge and NHS Blood and Transplant

Deininger, Tyner and Solary. Nat Rev Cancer 2017

5-155

ageing

premalignancy

malignancy

The role of the HSC

microenvironment

during:

A continuum in the myeloid malignancies

Two anatomical HSC niches in the bone marrow: central and endosteal

Mendez-Ferrer S et al. Nat Rev Cancer 2020

Nes-GFPhi NG2+

BMSC

HSC

increased:central niche

noradrenergic fibres

capillaries with Nes-GFP+ BMSCs

HSC proliferation

myelopoiesis

EC

reduced:endosteal niche

transition zone vessels and arterioles

HSC quiescence and self-renewal

resistance to genotoxic stress

regenerative haematopoiesis

lymphopoiesis

oste

ob

las

ts

sinusoid

sinusoid

transition zone

vesseleeee

arterioleeeeeeleleleeeaaaaaararaaaaaaa

capillary

Nes-GFP+

BMSC

osteo

blast

s

HSCH

EC

capillary

capillary

Nes-GFP+

BMSC

Increased central niches and reduced endosteal niches promote myeloid expansion during ageing

Ho Y-H et al. 2019 Cell Stem Cell 25:407-18

adult old

Reduction of endosteal niches and expansion of non-endosteal niches during normal ageing

CD31

EMCN youn

gold

0

10

20

30

EM

NC

hi C

D31

hi v

essels

(rela

tive t

o b

one length

) **

TZVs sinusoids

youn

gold

0.0

0.1

0.2

0.3

CD

31

lo E

MC

Nlo s

inusoid

s

(rela

tive t

o B

M a

rea)

youn

gold

0

5

10

15

20

25

CD

31

hi E

MC

N- capill

aries

(x10

-6;

rela

tive t

o B

M a

rea) ***

youn

gold

0

2

4

6

8

CD

31

hi E

MC

N- art

eriole

s

(x10

-6;

rela

tive t

o B

M a

rea) *

arterioles capillaries


adult old

Nes-GFP

CD31

EMCN

adult old

0.0

0.5

1.0

1.5

2.0

Nes-G

FP

+ c

ells

(% C

D45

- C

D31

- Ter1

19

- cells

)

e e n-e n-e

*

BMSCs

TH

Nes-GFP

CD31

TH

Contraction of endosteal niches and expansion of non-endosteal niches during normal ageing


Increased noradrenergic innervation of BM promotes MKpoiesis during ageing

Nes-GFP

CD31

TH

oldadult

youn

gol

d0.0

0.5

1.0

1.5

2.0

tyro

sin

e h

ydro

xyla

se +

are

a (

norm

alis

ed) *

youn

gol

d0

1

2

3**

skull tibia ****

0

500

1000

1500

2000

2500

pla

tele

ts p

er

ml o

f blo

od

(x10

4)

WT WT DKO DKO

oldadult

b2-b3-AR

** **

Pla

tele

ts p

er

ml o

f b

loo

d

(x1

06)

oldadult


b2-AR triggers IL6 release by the microenvironment to drive MKpoiesis during ageing

0

5

10

15

20

25

CD

41

+ c

ells

(%

LS

K)

WT b2

primary

**

AR KO

CD41+LS

K

0

5

10

15

donor

WT b2

recipient

WT b2

0

1 0

2 0

3 0

CD

41

+c

ell

s

(% C

D4

5.1

+L

SK

)

***

vehicle

b2-AR ag.

CD6

1

IL6 KO

CD41 CD42

DAPI

WT b2-AR KO

0

50

100

150

b3WT AR KO b2

***

CD

41

+C

D4

2+

meg

a-

ka

ryocyte

s p

er

mm

2

0

20

40

60

80

100

120

CD41

+ CD42

+ ce

lls p

er m

m2 *

WT b2 KO IL6 KO

0

5

10

15

20

25

IL-6

(pg p

er

ml) in B

ME

CF

WT b2AR KO

*

0

200

400

600

800

1000

1200

CD

41

+ L

SK

cells

AR ag. b2- b2-

IL6+/+ IL6-/-

**

ns

IL6 protein IL6 mRNA myeloid culture

+ + --PKAi - - + +

*ns

0

1

2

3

IL6 m

RN

A (

fold

)

b2-AR ag.


Reduced b3-AR-NO expands central niches and increases MKpoiesis during ageing

0

20

40

60

CD150lo/-

CD41- HSCs (%)

0

20

40

60

CD150lo/-

CD41- HSCs (%)

0

20

40

60

80

CD

150

lo/-C

D41

- H

SC

s (

%)

0

20

40

60

80

CD

150

lo/-C

D41

- H

SC

s (

%)

*

b3AR ag - b3-

DMSO L-VINO

0

200

400

600

800

Vw

f-eG

FP

- H

SC

s

*

WT b3 KO0

2

4

6

8

nitra

tes (

mM

) in

BM

EC

F

*

0.0

0.5

1.0

1.5

Nos1 m

RN

A (

fold

)

*

WT b3 KOWT b3 KO WT b3 KO WT b3 KO

primary recipient donor

p = 0.05

WT β3 Nos10

5

10

15

20

25

transitio

n z

one

vessels

(norm

aliz

ed) ***

p = 0.05

KO WT β3 Nos10

3

6

9

12

capill

aries (

norm

aliz

ed)

*****

KO 0

200

400

600

800

1000

1200

PLA

pla

tele

ts p

er

mm

3(x

10

3)

0

10

20

30

40

50

60

70

80

90

LYM MON NEU

WB

Cs (

%)

*

*

*

*

WT Nos1-/-

Eriksson, M. et al. Nature 423, 293-298 (2003)

De Sandre-Giovannoli, A. et al. Science 300, 2055 (2003)

Premature ageing in Hutchinson-Gilford progeria syndrome

Lymphoid deficiency and myeloid skewing during premature ageing …

***

0

200

400

600

800

1000

1200

1400

1600

PLA

pla

tele

ts p

er

mm

3(x

10

3)

**

***

***

0

10

20

30

40

50

60

70

80

NEU LYM MON EOS BAS

WB

Cs (

%)

WBCs

WT

progeroid

0

5

10

15

20

25

CD

3+ c

ells

(%)

0

20

40

60

B220

+ c

ells

(% )

0

10

20

30

CD

11b

+ c

ells

(%)

****

0

200

400

600

800

1000

1200

1400

1600

PLA

pla

tele

ts p

er

mm

3(x

10

3)

0

10

20

30

40

50

60

70

80

NEU LYM MON EOS BAS

WB

Cs (

%)

... is NOT haematopoietic cell-autonomous

0

10

20

30

40

CD

3+ c

ells

(% d

onor

cells

)

0

10

20

30

40

50

B220

+ c

ells

(% d

onor

cells

)

0

10

20

30

CD

11b

+ c

ells

(% d

onor

cells

)

myeloid skewing increased platelets


WT progeroid

0

5

10

15

20

25

IL1β

(pg p

er

ml) in

BM

EC

F

0

20

40

60

80

IL1

a (pg p

er

ml) in B

ME

CF *

0

2

4

6

IL6

(pg p

er

ml) in B

ME

CF *

0

2

4

6

8

IFNγ (

pg p

er

ml) in B

ME

CF **

p = 0.06

0.0

0.5

1.0

1.5

2.0

IL3

(pg p

er

ml) in B

ME

CF

p = 0.09

Similar myelopoietic cytokines increase in BM during physiological/premature ageing

physiological

aging

progeria


β3-AR agonist improves lineage skewing and HSC numbers in progeria

WT progeroid, vehicle progeroid, b3-AR agonist


Summary (I): premature/physiological niche ageing promotes myeloid expansion

IL6

β2-AR

NO

distance

non-endosteal niches

Mk

LT-HSC

myeloid skewing

megakaryocyte

differentiation

β3-AR

TZVs

endosteal niches

Ly-HSC My-HSC

β3-AR signalling β2-AR signalling

capillary

arteriole

sinusoid

BMSC

MkP

NOS1

X

Hou F-H & Ho K-Y

▪ Reduction of endosteal BM and expansionofnon-endosteal BM occurs with age

▪ β2-AR overriding β3-AR promotesmyeloid expansion during normal aging

▪ Premature HSC aging inprogeria can beimproved by targeting themicroenvironment

▪ β2/β3-ARs exhibit opposite and niche-dependent regulation of myelopoiesis


Deininger, Tyner and Solary. Nat Rev Cancer 2017

5-155

ageing

premalignancy

malignancy

The role of the HSC

microenvironment

during:

A continuum in the myeloid malignancies

Two non-mutually exclusive contributions for haematopoietic niches to leukaemia:

1. The acquisition of mutations or functional alterations by

niche cells that predispose for malignancy development

2. Niche remodelling by transformed haematopoietic cells

that facilitates disease manifestation and/or progression

The transformed niche

RARg deficiency in microenvironment causes MPN

Walkley CR, ..., Purton LE. Cell 129(6):1097-1110 (2007)

Combined retinoblastoma deletion in microenvironment

and myeloid cells causes MPN

Walkley CR, ..., Orkin SH. Cell 129(6):1097-1110 (2007)

Microenvironmental Notch inhibition causes

reversible, non-transplantable MPN

Kim YW, ..., Kong YY. Blood 112(12):4628-38 (2008)

Endothelial-specific Rbpj deletion can induce MPN-like disease

Wang L, Carlesso N et al. Cell Stem Cell 15(1):51-65 (2014)

Ptpn11E76K/+ mutation in nestin+ BMSPs aberrantly

activates neighbouring WT HSCs, inducing JMML

Dong L, ..., Qu CK. Nature 2016;539:304-8

Reduced Sipa1 expression in BM microenvironment

triggers MPN/MDS-like disease

granulocytes

Xiao P, ..., Qian H. Blood Adv. 2018;2:534-48

BMSCs

mR

NA

Niche alterations in mouse models predispose to haematological malignancies


Two non-mutually exclusive contributions for haematopoietic niches to leukaemia:

1. The acquisition of mutations or functional alterations by

niche cells that predispose for malignancy development

2. Niche remodelling by transformed haematopoietic cells

that facilitates disease manifestation and/or progression

The transformed niche

2.1. Niche stromal cell reprogramming

2.2. Inflammation

2.3. Hypoxia and Angiogenesis

2.4. Activation of survival pathways

2.5. Protection from excessive ROS (metabolic reprogramming)

2.6. Immunosuppression

2.7. Therapeutic resistance

MPNs were initially thought to be solely driven by mutated HSCs

Mughal TI, ...,Van Etten RA. Leuk Lymphoma 57(7):1517-26 (2016)

control human MPN

NESTIN

Reduction of BM nestin+ cells in human and murine MPN

0

2

4

6

8

10

**

mR

NA

(ra

tio

)

Control MPN

NESTIN

0

500

1000

1500

2000

2500

**

C JAK2-

V617F

BM Nestin-GFP+ cells

0

100

200

300

400

500

BM CFU-F

C JAK2-

V617F

**

JAK2-V617F

Control

Arranz L et al. Nature 2014;752:78-81

0

5

10

15

20

*Contro

l

(C)

JAK2-

V617F

Nestin

mR

NA

(fo

ld)

BM neuropathy precedes apoptosis of nestin+ cells in

MPN

Control

JAK2V617F

Nes-GFP GFA

P

TH

200 mm100 mm

Arranz L et al. Nature 2014;752:78-81

b3-AR agonist rescues nestin+ niches and improves myelofibrosis in mice and humans

Drexler B et al. Haematologica 2019Arranz L et al. Nature 2014; 512:78-81

β3-AR agonist

0

500

1000

1500

2000

2500

BM Nestin-GFP+ cells

*

veh β3 ag

vehicle b3-AR agonist before after

myelofibrosis nestin+ niches

Herlihy N, Harrison CN, McLornan DP 2019 Haematologica 104(4):639-641©2019 by Ferrata Storti Foundation

Exploitation of the neural-HSC niche axis to treat myeloproliferative neoplasms

BMSC-derived placental growth factor supports CML cells

Schmidtt T, ..., Carmeliet P. Cancer Cell 2011;19:740-53

Expansion of inflammatory osteoblasts in chronic

phase CML impairs normal haematopoiesis

Schepers K, ..., Passegué E. Cancer Cell 2013;13:285-99

Frisch BJ, ..., Calvi LM. Blood 2012;119:540-50

Functional osteoblast inhibition in blast crisis CML

Krause DS, ..., Scadden DT. Nat Med 19, 1513–7 (2013)

Parathormone signaling on osteoblasts has

opposite effects on CML and AML

CML AML

BM engraftment: Pre-LSC = HSC vs. LSC = GMP

Lane SW, ..., Williams DA. Blood 118(10):2849-56 (2011)

SNSMSC

OBsHSC KSL/prog

Lymphocytes

Myeloid cells

HEALTH

SNSMSC

HSC KSL/ProgLeukHSC

IL-1β

1

OBs

IL-6

3

KSL/Prog

OBs

LymphocytesMyeloid cells

IL-6

MPN

4

Model of stepwise microenvironment alterations in MPN

Leuk prog

LeukHSC

2

Myeloid cells

Mendez-Ferrer S et al. Cancer Cell 2015;27(5):611-3

Kfoury Y and Scadden DT. Cell Stem Cell 2015

Different waves of BMSCs with distinct functions

Mesenchymal stem cell (MSC)

Adipoblast

Skeletal precursor

Preosteoblast

Osterix

Fibroblastic

reticular cells

Stromal lineages

PPARγ

Haematopoietic

lineages

LT

HSC

MPP

MEPGMP

CMP

CLP

proTproB

A hierarchical regulation in the bone marrow?

Osteoblast

ST

HSC

nestin

Lepr-cre

Bone and cartilage are derived from lineage-restricted progenitors

(and not BMSCs)

Chan CKF et al. Cell 160:285-298 (2015)

Nestin+ cells do not differentiate into osteoblasts/fibroblasts in MPN

Nestin-creERT2;RCE JAK2V617FControl

200 µm

Arranz L, ..., Mendez-Ferrer S. Nature 2014;752:78-81

Nat Cell Biol

2017

Ding L, ..., Morrison SJ. Nature 481:457-62 (2012)

Cell

Stem

Cell

2014

Osteocyte

Preosteoblast Adipoblas

t

Adipocyte

Chondroblast

Chondrocyte

Skeletal

precursor

Mesenchymal

stem cell

(MSC)

Fibroblastic

reticular cells

Osteoblast

Lepr-cre targeted cells (postnatal BM expression)

Prx1-cre targeted cells (prenatal BM expression)

HSC

niche

factors

Tie2-cre

Constitutive Cre lines might be convenient to obtain strong

phenotypes but are not very informative of the cell of origin

Endothelial cell

Reduction of nestin+ cells and expansion of Gli1+ cells in PMF

PMF: nestin+ cells Gli1+ cells Gli1+ cells are Lepr-, but some are nestin+

Schneider RK, ..., Kramann R. Cell Stem Cell 20:785-800 (2017)

Chronic treatment with Gli1/2 inhibitor improves myelofibrosis in mice

Schneider RK, ..., Kramann R. Cell Stem Cell 20:785-800 (2017)

Monocyte-derived fibrocytes may originate myelofibroblasts

PMF: BMSCs Monocyte-derived fibrocytes

Chronic treatment with the fibrocyte inhibitor serum amyloid P (pentraxin-2)

improves myelofibrosis and survival in mice

Verstovsek S et al. J Exp Med 2016;213(9):1723-40

Monocyte-derived fibrocytes may originate myelofibroblasts

Maekawa T, ..., Kimura F. Leukemia 2017;31:2709-16

Angiocrine support of normal and malignant stem cells

Butler JM, Kobayashi H and Rafii S. Nat Rev Cancer 10(2):138-46 (2010)

Increased BM angiogenesis in MPNs

Boveri E, ..., Passamonti F. Br J Haematol 140(2):162-8 (2008)

PV ET

Pre-fibrotic

PMF

Fibrotic

PMF

Autocrine and paracrine effects of angiogenic factors

Kampen KR, ..., de Bont ES. Cell Mol Life Sci 70(8):1307-17 (2013)

BM microenvironment is hypoxic in myeloid malignancies

Rieger C and Fiegl M.Exp Hematol 44:578-82 (2016)

Hif1a loss accelerates FLT3ITD-induced MPN

Velasco-Hernandez T, Cammenga J et al. Leukemia 29:2366-74 (2015)

Hif1a and Hif2a support LSC survival in CML and AML

Zhang H et al. Blood 119:2595-2607

(2012)

Roualt-Pierre K, Bonnet D et al.

Cell Stem Cell 13(5):549-63 (2013)

Microenvironment alterations early during MPN development

Korn C and Mendez-Ferrer S. Blood 2017; 129(7):811-822

The BM microenvironment at intermediate MPN stage


The BM microenvironment at late MPN stage


BM niche remodelling favours disease progression in haematological malignancies


Contributions of the BM niche to survival and chemoresistance of malignant haematopoietic cells


External funding:

Collaborators Institution

Tony Green

University of CambridgeBrian Huntly

Adrien Hallou

Ben Simons

Alex Theoccarides Zurich University Hospital

Jürg Schwaller

Basel University HospitalRadek Skoda

Alexandar Tzankov

Claus Nerlov Oxford University

Cristina Lo Celso Imperial College, London

Dominique Bonnet Crick Institute, London

WT-MRC Cambridge Stem Cell Institute

Current lab members

Claire Fielding

Elodie Grockowiak

Ya-Hsuan Ho

Giuditta Corbizi Fattori

Antonio Rodriguez

Zijian Fang

Stephen Gadomski

Jun Zhang

Thomas McKerrell

Jane Cook

Acknowledgements

Previous lab members

Dorian Forte

Claudia Korn

Justyna Rak

Andres Garcia

Maria Garcia-Fernandez

Joan Isern

Lorena Arranz

Daniel Martin-Perez

Sandra Martin

Abel Sanchez-Aguilera

Carlos Lopez

The bone marrow microenvironment in myeloid malignancies€¦ · 12 c a p i l l a r i e s (n o r m...

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