What is hematopoiesis ? Neutrophils : 24 hrs Platelets : 6-7 days Red cells : 120 days. Constant renewal of a pool of differentiated cells : red cells /kg/day (10 12 cells per day) Thus, to maintain homeostasis, the system must have the capacity for self renewal The 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 Differentiation Progenitors Stem cell Self-renewal Symmetric or asymmetric divisions

Sequential events leading to mature blood cells Hematopoietic 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 progenitors

Phenotype and hematopoietic hierarchy

Hematopoietic stem cells are quiescent (non cycling)

Fibroblasts Osteoblasts ECM(OPN) Adipocytes Vascular niche NormoxiaCytokines (Tpo, SCF, HGF, FGF4…) Chimiokines (Il-8, SDF-1..) Hypoxia Ca ++ Proteases (MMP9, CTK…) OsteoclastsCSM ESC HSC The bone marrow hematopoietic niches Endosteal niche CAR cells Ang-1/Tie2 Wnt/ β -caténin Jagged-1/Notch..

HSC Progenitors Precursors Cytokines TPO SCF G-CSF Interferon Morphogenes Cellular interactions BMP/TGF  Notch Wnt Shgh Chimiokines SDF1 Quiescence Self-renewal Differentiation Homing Proliferation Differentiation Cell functions

HSC Progenitors Precursors Quiescence Self-renewal Differentiation Homing Proliferation Differentiation Cell functions 5x x10 6 HSC Average doubling time 45 days 15 mitosis Asymmetric/symmetric division : Extinction of stem cells (exhaustion, apoptosis)

Niche (Competition and clonal dominance) Differentiation Asymmetric division Clonal dominance induced by symetrical mitosis Symmetric division Asymmetric division

The differentiation hierarchy of the hematopoietic system. Haeno H et al. PNAS 2009;106:

Cancer stem cell/leukemic stem cell Tumor is arranged as a hierarchy similar to normal hematopoietic tissue Block terminal differentiation Mature cells CD34+/ CD38- CLP CMP HSC Leukemic stem cell Leukemogenic events Bulk leukaemia cells Leukemia Hematopoietic reconstitution QuiescencePhenotype Niche dependency ? HSC Progenitors Precursors LSC Progenitors Precursors AML LSC Progenitors Precursors MDS LSC Progenitors MPD Precursors

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

WHO classification of MPDs Essential Thrombocythemia (ET) CML Classical MPDs Rare and unclassified MPDs Polycythemia vera (PV) Myelofibrosis (PMF) Bcr-abl PDGFRa FGFR1 Kit

BFU-E CFU - E B cell B cell PMEo PMB MonoPMN CFU-MK Pre-BPre-TRBCMacrophage PLT T cell T cell DC NK NK CFU-GM CFU-Eo CFU-baso MPDs B cell B cell PEo PB MonoPN CFU-MK CFU-baso Pre-B Pre-T CFU-Eo CFU-GM BFU-E CFU-ERBCPRECURSORS Mature cells Macrophage PLT HSC T cell T cell DC NK NK PROGENITORS Normal hematopoiesis MPDs are clonal diseases involving HSC leading to increase blood cell production

BFU-E CFU - E B cell B cell PMEo PMB MonoPMN CFU-MK Pre-BPre-TRBCMacrophage PLT T cell T cell DC NK NK CFU-GM CFU-Eo CFU-baso B cell B cell PEo PB MonoPN CFU-MK CFU-baso Pre-B Pre-T CFU-Eo CFU-GM BFU-E CFU-ERBCPRECURSORS Mature cells Macrophage PLT HSC T cell T cell DC NK NK PROGENITORS Clonal dominance of the MPD clones (log-log age incidence curve) Up to 60 years 1/1x10 6 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 50% BCS C-term N-term JH1 JH2 Kinase domain Pseudokinase domain Cytokine receptor Interacting domain V617F FERM domain SH2 K539L JAK2V617F Baxter, The Lancet 2005 James, Nature 2005 Kralovics, NEJM 2005 Levine, Cancer Cell 2005 JAK2 exon 12 Scott, NEJM 2007 MPL W515 Pickman, PLoS med 2006

A proposed structure of JAK2V617F Lee et al. Cancer 15, 1692, 2009

PV2 heterozygous The mutation is homozygous in 30% of the PV patients by duplication of the mutated allele N AAAANCG AAAACCG AAAAACG PV1 homozygous FISH JAK2: the two alleles are present This homozygosity correlates with 9pLOH (Kralovics et al 2005) and is related to a mitotic recombination ACGCCGN T cells ACGCCGT Granulocytes

AAAA N CG AAAA A CG Heterozygous ? Homozygous ? Homozygous Allele ratio from blood leukocyte DNA in PV and ET patients ET PV

Clonal dominance in MPD clones BFU-E CFU - E B cell B cell PMEo PMB MonoPMN CFU-MK Pre-BPre-TRBCMacrophage PLT T cell T cell DC NK NK CFU-GM CFU-Eo CFU-baso BFU-E B cell B cell PMEo PMB MonoPMN CFU-MK Pre-BPre-TRBCMacrophage PLT T cell T cell DC NK NK CFU-GM CFU-Eo CFU-baso CFU - E ETPV

ET PV (heterozygous profile) PV (homozygous profile) Size of the JAK2 V617F clone CD34 + CD38 - CD34 + CD38 + ErythroblastsGranulocytes Clonal amplification Commited Progenitors (BFU-E, CFU-GM) Precursor cells Erythroblasts Granulocytes B cells, NK Lympho- Myeloid progenitors CD34 + /38 - ThrombocytosisErythrocytosis Homo. JAK2 V617F Normal marrow PV homozygous PV heterozygous 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 q Mb MPD01 MPD04 MPD05 TET2 5'3' Mb Mb 325 Kb deletion LOH SNP Array MPD05 MPD02 MPD01 Copy neutral LOH Discovery of a cooperative genetic events

TET2 loss of function:increase in HSC/progenitors or both ? TET2 JAK2 VF CD19 CD56 CD1 5 NOD-SCID HSC lympho-myeloid progenitor cell lymphoid progenitor cell MEP BFU-E CFU-E CFU-MK CFU-GM CFU-M CFU-G RBCsplateletsGranulocytes T cell B cell NK Erythroblast Megakaryocyte Monocytes CD15 CD19 CD56 late onset of clonality Proliferative advantage Early onset of clonality Facilitates transformation by increasing cells targetted by oncogenic hits

Modeling cancer disease A model of cancer initiation Haeno H et al. PNAS 2009;106:

The probability of cancer initiation along the four evolutionary trajectories Haeno H et al. PNAS 2009;106:

The dominant trajectory to MPN-initiating cells. Haeno H et al. PNAS 2009;106:

Vickers, M. A. Blood 2007;110: 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 ( /cell/year) - Loss of HSC through differentiation ( the rate of symmetrical divisions) - 60%-99% of all clones are extinguished

Murine models to model the disease (KI Mice) E12 TTC (F617) E13m E14 KI/Cre CMV promoter Cre VAV promoter Cre SCL promoter 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 Bcr-abl PDGFRa FGFR1 Kit Modeling response to targeted therapies The 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 cells The 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-E CFU - E B cell B cell PMEo PMB MonoPMN CFU-MK Pre-BPre-T RBC Macrophage PLT T cell T cell DC NK NK CFU-GM CFU-Eo CFU-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 Bernard V é ronique Della Valle Roland Berger Ludwig Institute for Cancer Research (Brussels) Stefan Constantinescu INSERM U1009 Villejuif Eric Solary François Delhommeau Nicole Casadevall Stéphane Giraudier Rodolphe Besancenot Jean-Pierre Le Couédic Isabelle Plo Jean Luc Villeval