What is hematopoiesis ?

Neutrophils : 24 hrs Platelets : 6-7 days Red cells : 120 days.

Constant renewal of a pool of differentiated cells: 2. 109 red cells /kg/day(1012 cells per day)

Thus, to maintain homeostasis, the system must have the capacity for self renewalThe paradox is that the stem cell at the top of the system is quiescent

Hematopoiesis is a term describing the formation and development of blood cells

Cells of the blood are constantly being lost or destroyed

Blood harbors many different kinds of cells

Differentiation

Stem cell

Progenitors

Self-renewal

What is a stem cell

Differentiation

Stem cell

Progenitors

Self-renewal

Asymmetric division

Symmetric divisions

Progenitors

Stem cell

DifferentiationProgenitors

Stem cell

Self-renewal Self-renewal

Symmetric or asymmetric divisions

Sequential events leading to mature blood cells

Hematopoietic stem cellsHematopoietic stem cells(rare and undifferentiated)

Mature cells -with specific function (i.e. O2 transport) -represent most of the cells

Many intermediate states with varying degree of

proliferation and differentiation

Hematopoietic cells can be divided into three cellular compartments

Hematopoietic progenitorsHematopoietic progenitors

Phenotype and hematopoietic hierarchy

Hematopoietic stem cells are quiescent (non cycling)

FibroblastsFibroblasts

OsteoblastsOsteoblasts

ECMECM(OPN)(OPN)

AdipocytesAdipocytes

Vascular nicheVascular niche NormoxiaNormoxia

CytokinesCytokines(Tpo, SCF, HGF, FGF4…(Tpo, SCF, HGF, FGF4…))

ChimiokinesChimiokines(Il-8, SDF-1..)(Il-8, SDF-1..)

HypoxiaHypoxiaCaCa++++

ProteasesProteases(MMP9, CTK…)(MMP9, CTK…)

OsteoclastsOsteoclastsCSMCSM

ESCESC

HSHSCC

The bone marrow hematopoietic nichesThe bone marrow hematopoietic niches

Endosteal nicheEndosteal niche

CAR cellsCAR cells

Ang-1/Tie2Ang-1/Tie2Wnt/Wnt/ββ-caténin -caténin Jagged-1/Notch..Jagged-1/Notch..

HSCHSC

ProgenitorsProgenitors

PrecursorsPrecursors

Cytokines

TPOSCF

G-CSFInterferon

MorphogenesCellular interactions

BMP/TGFNotchWntShgh

ChimiokinesSDF1

QuiescenceSelf-renewal

DifferentiationHoming

ProliferationDifferentiation

DifferentiationCell functions

HSCHSC

ProgenitorsProgenitors

PrecursorsPrecursors

QuiescenceSelf-renewal

DifferentiationHoming

ProliferationDifferentiation

DifferentiationCell functions

5x105-3x106 HSC

Average doubling time 45 days

15 mitosis

Asymmetric/symmetric division: 0.5-3

Extinction of stem cells(exhaustion, apoptosis)

Niche(Competition and clonal dominance)

Differentiation

Asymmetric division

Clonal dominance induced by symetrical mitosis

Symmetric division Symmetric division

Symmetric division

Symmetric division

Asymmetric division

Asymmetric division

The differentiation hierarchy of the hematopoietic system.

Haeno H et al. PNAS 2009;106:16616-16621

Cancer stem cell/leukemic stem cellTumor is arranged as a hierarchy similar to normal hematopoietic tissue

Block terminaldifferentiation Mature cells

CD34+/ CD38-

CLP CMP

HSCLeukemic stem cell Leukemogenic

events

Bulk leukaemiacells

Leukemia Hematopoietic reconstitution

QuiescenceQuiescencePhenotypePhenotypeNiche Niche dependency ?dependency ?

HSCHSC

ProgenitorsProgenitors

PrecursorsPrecursors

LSCLSC

ProgenitorsProgenitors

PrecursorsPrecursors

AML

LSCLSC

ProgenitorsProgenitors

PrecursorsPrecursors

MDS

LSCLSC

ProgenitorsProgenitors

MPD

PrecursorsPrecursors

The leukemic initiating genetic event can occur in an HSC or a progenitor by reprogrammation into a HSC

Oncogenic eventOncogenic event

Self-renewal

WHO classification of MPDs

Essential Thrombocythemia

(ET)

CML Classical MPDs Rare and unclassified MPDs

Polycythemia vera (PV)

Myelofibrosis(PMF)

Bcr-abl PDGFRaFGFR1Kit

BFU-EBFU-E

CFUCFU--EE B cell B cell

PMEoPMEo PMBPMBMonoMonoPMNPMN

CFU-MKCFU-MK Pre-BPre-B Pre-TPre-T

RBCRBC

MacrophageMacrophage

PLTPLT

T cell T cell

DCDC

NK NK

CFU-GMCFU-GMCFU-EoCFU-EoCFU-basoCFU-baso

MPDs

B cell B cell PEoPEo PBPBMonoMonoPNPN

CFU-MKCFU-MK CFU-basoCFU-basoPre-BPre-B Pre-TPre-TCFU-EoCFU-EoCFU-GMCFU-GMBFU-EBFU-E

CFU-ECFU-E

RBCRBC

PRECURSORSPRECURSORS

Mature cellsMature cells

MacrophageMacrophage

PLTPLT

HSCHSC

T cell T cell

DCDC

NK NK

PROGENITORSPROGENITORS

Normal hematopoiesis

MPDs are clonal diseases involving HSC leading to increase blood cell production

BFU-EBFU-E

CFUCFU--EE B cell B cell

PMEoPMEo PMBPMBMonoMonoPMNPMN

CFU-MKCFU-MK Pre-BPre-B Pre-TPre-T

RBCRBC

MacrophageMacrophage

PLTPLT

T cell T cell

DCDC

NK NK

CFU-GMCFU-GMCFU-EoCFU-EoCFU-basoCFU-baso

B cell B cell PEoPEo PBPBMonoMonoPNPN

CFU-MKCFU-MK CFU-basoCFU-basoPre-BPre-B Pre-TPre-TCFU-EoCFU-EoCFU-GMCFU-GMBFU-EBFU-E

CFU-ECFU-E

RBCRBC

PRECURSORSPRECURSORS

Mature cellsMature cells

MacrophageMacrophage

PLTPLT

HSCHSC

T cell T cell

DCDC

NK NK

PROGENITORSPROGENITORS

Clonal dominance of the MPD clones(log-log age incidence curve)

Up to 60 years1/1x106 1x10-3 to 100%

Molecular events and classical MPDs

PMF

45% 25%

atypical MPDs

50%

5%MPL W515

PV

95%

JAK2 Exon 12

JAK2V617F

ET

30%65%

MPL W515

3%-5%

JAK2V617F

JAK2V617F

50%

BCS

C-termN-termJH1JH2

Kinase domainPseudokinasedomain

Cytokine receptorInteracting domain

V617F

FERM domainSH2

K539L

JAK2V617F Baxter, The Lancet 2005 James, Nature 2005Kralovics, NEJM 2005Levine, Cancer Cell 2005JAK2 exon 12Scott, NEJM 2007MPL W515Pickman, PLoS med 2006

A proposed structure of JAK2V617FLee et al. Cancer 15, 1692, 2009

A proposed structure of JAK2V617FLee et al. Cancer 15, 1692, 2009

PV2heterozygous

The mutation is homozygous in 30% of the PV patients by duplication of the mutated allele

N

A A AAN CG

A A AAC CG

A A AAA CG

PV1homozygous

FISH JAK2: the two allelesare present

This homozygosity correlates with 9pLOH (Kralovics et al 2005) and is related to a mitotic recombination

AC GCC GN

T cells

AC GCC GT

Granulocytes

A A AAN CG

A A AAA CG

Heterozygous ?Homozygous ?

Homozygous

Allele ratio from blood leukocyte DNAin PV and ET patients

ET

PV

Clonal dominance in MPD clones

BFU-EBFU-E

CFUCFU--EE B cell B cell

PMEoPMEo PMBPMBMonoMonoPMNPMN

CFU-MKCFU-MK Pre-BPre-B Pre-TPre-T

RBCRBC

MacrophageMacrophage

PLTPLT

T cell T cell

DCDC

NK NK

CFU-GMCFU-GMCFU-EoCFU-EoCFU-basoCFU-baso BFU-EBFU-E

B cell B cell

PMEoPMEo PMBPMBMonoMonoPMNPMN

CFU-MKCFU-MK Pre-BPre-B Pre-TPre-T

RBCRBC

MacrophageMacrophage

PLTPLT

T cell T cell

DCDC

NK NK

CFU-GMCFU-GMCFU-EoCFU-EoCFU-basoCFU-baso

CFUCFU--EE

ET PV

ET

PV (heterozygous profile)

PV (homozygous profile)

1.0

0.6

0.0

0.2

0.8

0.4

Siz

e of

th

e JA

K2

V61

7F c

lon

e

CD34+CD38- CD34+CD38+ Erythroblasts Granulocytes

Clonal amplification

CommitedProgenitors

(BFU-E, CFU-GM)

Precursor cellsErythroblastsGranulocytesB cells, NK

Lympho-Myeloid progenitors

CD34+/38-

Thrombocytosis Erythrocytosis Erythrocytosis

Homo.JAK2V617F

Normalmarrow

PVhomozygous

PVheterozygous

ET(heterozygous)

PMF

Fibrosis

Is JAK2 signaling sufficient to induce a clonal dominance at the level of HSC ?(Requirement for other mutations such as TET2 ? )

Chromosome 4 q24

107.33 Mb

MPD01

MPD04

MPD05

TET25' 3'

106.11 Mb 106.35 Mb

325 Kb deletion

LOH

SNP Array

MPD05

MPD02

MPD01

Copy neutralLOHCopy neutralLOH

Discovery of a cooperative genetic events

TET2 loss of function:increase in HSC/progenitors or both ?

TET2

JAK2VF

JAK2VF

JAK2VF

JAK2VF

100 101 102 103 104

CD15 FITC100 101 102 103 104

CD15 FITC

CD

19

CD

56

CD15

CD15

NOD-SCID

HSC

lympho-myeloid progenitor cell

lymphoid progenitor cell

MEP

BFU-E

CFU-E

CFU-MK

CFU-GM

CFU-MCFU-G

RBCs platelets Granulocytes T cell

B cell

NK

Erythroblast

Megakaryocyte

Monocytes

CD15 CD15

CD

19

CD

56

late onset of clonality

Proliferative advantageEarly onset of clonalityFacilitates transformation by increasing cells targetted by oncogenic hits

Modeling cancer disease A model of cancer initiation

Haeno H et al. PNAS 2009;106:16616-16621

The probability of cancer initiation along the four evolutionary trajectories

Haeno H et al. PNAS 2009;106:16616-16621

The dominant trajectory to MPN-initiating cells.

Haeno H et al. PNAS 2009;106:16616-16621

Vickers, M. A. Blood 2007;110:1675-1680

Illustrative simulations showing age-specific incidence curves predicted from single mutation conferring an "exponential phenotype

Conclusions- Minimum 18 V617F HSC to get a phenotype- Symmetrical divisions (0.2-0.4/cell/year)- Loss of HSC through differentiation (0.8-0.95 the rate of symmetrical divisions)- 60%-99% of all clones are extinguished

Murine models to model the disease(KI Mice)

E12

TTC (F617)

E13m

KI/Cre

CMVpromoter Cre

VAVpromoter Cre

SCLpromoter CreERt

embryonic / Ubiquitous

Expression

embryonic / hematopoietic

Hematopoietic / inducible(tamoxifen)

Promoter

WHO classification of MPDs

Essential Thrombocythemia

(ET)

Classical MPDs Rare and unclassified MPDs

Polycythemia vera (PV)

Myelofibrosis(PMF)

CML

PDGFRaFGFR1Kit

Modeling response to targeted therapiesThe model of CML

• CML is caused by a fusion protein BCR-ABL

• BCR-ABL can be targeted by Imatinib (a TK inhibitor)

• Clinical success but does not eradicate the disease

• Resistance to treatment can also appear

Response to therapy and modeling(Michor et al. Nature 2005)

Two curves:The first corresponds to the killing of maturing cellsThe second to the effects on more primitive cells, Progenitor or stem cell ?

Need for mathematical models to improve the treatment

Exhaustion of HSC through proliferation(the exemple of interferon)

Trump et al.

Polycythemia Vera and pegylated Interferon alpha (Kiladjian et al. Blood, 2008)

Effect on a leukemic stem cell ?

BFU-EBFU-E

CFUCFU--EE B cell B cell

PMEoPMEo PMBPMBMonoMonoPMNPMN

CFU-MKCFU-MK Pre-BPre-B Pre-TPre-T

RBCRBC

MacrophageMacrophage

PLTPLT

T cell T cell

DCDC

NK NK

CFU-GMCFU-GMCFU-EoCFU-EoCFU-basoCFU-baso

1x10-3 to 100%

Minimum 18 V617F HSC to get a phenotype

Drugs which would activate HSC(Interferon ?)

Drugs which would kill specifically the proliferating

mutated cells(specificic JAK2 inhibitor)

Conclusions

• Modeling of normal hematopoiesis may be a powerful tool to predict complex biologic processes that will be validated by experimental procedures

• Modeling of malignant hematopoiesis may help to understand the clonal dominance and the cooperative effects of several genetic alterations

• Modeling might be a powerful tool to prospectively in vivo monitor the response to a drug and a combination of drugs

• MPD appear to be disorders whose pathogenesis and treatment might be greatly improved by such an approach

Acknowledgments

INSERM U985

Villejuif

Olivier BernardVéronique Della Valle

Roland Berger

Ludwig Institute for Cancer Research (Brussels)

Stefan Constantinescu

INSERM U1009Villejuif

Eric Solary

François Delhommeau

Nicole Casadevall

Stéphane Giraudier

Rodolphe Besancenot

Jean-Pierre Le Couédic

Isabelle Plo

Jean Luc Villeval