Myeloproliferative neoplasms: Are ET, PV and PMF the same or - PowerPoint PPT Presentation

Myeloproliferative neoplasms: Are ET, PV and PMF the same or different disorders ?

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

MPNs are clonal diseases involving the HSC leading to increase blood cell production Normal hematopoiesis MPNs HSC HSC PROGENITORS PROGENITORS CFU-GM CFU-GM CFU-Eo CFU-Eo CFU-MK CFU-MK BFU-E BFU-E Pre-B Pre-B Pre-T Pre-T BFU-E BFU-E CFU-baso CFU-baso CFU-basoPre-B Pre-B Pre-T CFU-Eo CFU-Eo CFU-MK CFU-MK CFU-baso Pre-T CFU-GM CFU-GM PRECURSORS PRECURSORS CFU-E CFU-E CFU-E CFU-E B cell B cell NK NK T cell T cell PN PN Mono Mono RBC RBC PEo PB B cell B cell T cell T cell PEo PB NK NK PLT PLT Mature cells Mature cells Macrophage Macrophage DC DC RBC RBC PMN Mono PMN Mono PMEo PMEo PLT PLT PMB PMB Macrophage Macrophage DC DC

Classification of MPNs has been based on clinical/biological criteria • Polycythemia Vera – MPN with a trilineage hyperplasia – Increase red cell mass (Hb > 18.5g/dl) • Primary myelofibrosis – Reticulin or collagen fibrosis – Megakaryocyte hyperplasia with dysplastic megakaryocytes and an increased granulocytic proliferation – No criteria of PV or other MPN • Essential Thrombocythemia – Sustained platelet count over 450x10 9 /L – Megakaryocyte hyperplasia with increased number of large megakaryocytes – No criteria of other MPN

Molecular events and classical MPNs JAK2 exon 12 JAK2 exon 12 JAK2V617F 3% 3% PV ET 50% 50% 95% 1% 1% LNK (exon 2) LNK (exon 2) 8% 8% JAK2V617F JAK2V617F ? JAK2V617F Baxter, The Lancet 2005 MPLW515 MPL W515 MPL W515 James, Nature 2005 Kralovics, NEJM 2005 Levine, Cancer Cell 2005 JAK2 exon 12 Scott, NEJM 2007 MPL W515 Pickman, PLoS med 2006 LNK exon 2 Oh, Blood 2010 CBL PMF 50% 50% Grand, Blood 2010 6% 6% 1% 1% LNK (exon 2) LNK (exon 2) c- CBL c- CBL 8% 8% MPL W515 MPL W515

PV is a homogeneous disorder related to a JAK2 constitutive activation requiring cytokine receptors JH7-JH5 JH4-JH3 JH2 JH1 JAK2 FERM SH2 pseudokinase kinase JAK2 fusion: CML, Peeters et al TEL JAK2 fusion: ALL, Peeters et al TEL JAK2 fusion: ALL, Lacronique et al TEL JAK2 fusion:T-cell lymphoma, PCM1 Adelaide et al PCM1 JAK2 fusion: CML, Bousquet et al JAK2 fusion: CML/ALL, Reiter et al PCM1 JAK2 fusion: MPD, Murati et al PCM1 FERM SH2 pseudokinase kinase V617F FERM SH2 pseudokinase kinase K539L

Cytokine receptors and JAK2 Cytokine receptors Cytokine receptors sharing β c subunit: IL-3R, sharing gp130 subunit: IL-6R, IL-5R, GM-CSFR IL-11R, OSMR, LIFR, Cytokine receptors sharing γ c subunit: IL-2R, IL4R IL-7R, IL-9R, IL-15R, IL21RInterferons & other Homodimeric cytokine receptors: EpoR, MPL, GCSFR Type II receptors: GHR, PrlR IFN α , IFN β , IFN γ , IL-10R, IL-19R, IL- 20R, IL-22R, IL- 24R, IL28R, IL29R JAK1/JAK2, TYK2 JAK1, JAK2/ JAK3 JAK2 JAK1, JAK2 and TYK2 (IFNs) JAK2 Type I Type II

Requirement of cytokine receptors V617F Y114A N-term C-term SH2 FERM domain JH2 JH1 Cytokine receptor Pseudokinase Kinase domain Interacting domain domain Wernig et al. Blood 2008

ET is a heterogeneous disorder related to a MPL constitutive activation pairs of cysteines JAK2V617F WSXWS Constitutive activation of MPL and EPOR JAK2V617F JAK2V617F box1 box2 MPL EpoR MPL mutants Ras P JAK JAK P 2 2 IRS2 T P Y Y P p o Sos Shc P P Y Y P STAT3/ Grb 2 5 P Y Y P STAT T487A P 3 Motif RWQFP S505N PI-3K JAK2/STAT essential for proliferation MPL Raf ∆5 MPL PKC ζ W515A/L/K WT WT T487A S505N TMPL MPL + Tpo ME Constitutive activation K of MPL STAT3 LNK mutants P P ER C-Akt STAT3 K Nucleus

ET is a heterogeneous clinical and biological disorder AL AML4 ET ET ET>PV>MF>AL ET ET AML ET ET>MF>AL Patient Plasma Serum P3 0 0 P5 0 0 P13 0 0 P32 0 0 Group 1 P34 0 0 P37 0 0 P52 0 0 P15 0 P41 0 P14 0 58 P22 0 78 P25 0 73 P44 0 31 P46 0 25 Group 2 P48 0 31 P49 0 336 P54 0 11 P59 0 25 P30 40 135 P33 100 177 P35 65 75 P38 50 163 P50 275 251 P55 17 153 Group 3 P56 37 100 P61 269 120 P62 10 221 P31 175 P39 32 P45 20

PMF is a heterogeneous disorder presently related to a very high MPL activation • In murine models, high MPL activation leads to a myelofibrosis: – Examples: over expression of TPO, JAK2V617F, MPLW515K/L/A, LNK Hyperleukocytosis with neutrophil polymorphs Giant platelets Erythro-myelemia Tear drop erythrocytes Normal spleen Splenomegaly Femurs Myelofibrosis Osteosclerosis • In murine models, all defects in MK maturation may lead to a myelofibrosis: Examples: GATA-1low, BACH1 overexpression • MK dysplasia implicated in the development of myelofibrosis • Possible role of neutrophils and monocytes in the myelofibrosis and inflammation

TPO Mpl 515 JAK2 V617F MEGAKARYOCYTE Implication of monocyte Implication of monocyte NF- κ B  TGF- β 1  IL-1 α Local activation Osteoblast Fibroblast Stromal cell proliferation stimulation proliferation  OPG Decrease of mature osteoclasts MARROW FIBROSIS OSTEOSCLEROSIS

High MPL activation normally induces senescence In normal MK differentiation, proliferation is limited by a senescence process Inflammatory secretome IL1 IL6 TGF β IL8 TPO IL10 JAK2 J A K 2 BRAF IL1 IL6 STATs TGF β PI3K IL8 RAF1 Cellular senescence IL10 MEK1/2 AKT ROS ERK1/2 DNA Repair Cell cycle arrest Inflammatory DNA damage p21 transcriptome EGR1 TF

This senescence process is inhibited in PMF (Requirement for several genetic or epigenetic abnormalities ?) D0 D3 D6 D9 pERK p21 Actin Normal Control p < 10 -2 p < 0.03 10 100 p21 expression (2 -∆Ct (p21-HPRT) ) SA-ß-Gal-positive cells (%) PMF p21 1 10 Actin 0.1 1 0.01 0.1 Healthy PMF Healthy donor PMF donor

High JAK2 signaling induces genetic instability and thus disease progression EoR V617F V617F S Cytoplasm S P JAK2 JAK2 O C Ras 1- High JAK2 signaling inhibits p53 function P P Y Y C Sos Shp-2 STAT5 I S Y P P Y 2- High JAK2 signaling stimulates HR Grb 2 Y Y Shp-1 P 3- High JAK2 signaling induces genetic instability PI-3K 4- High JAK2 signaling induces H3Y41 and LMO2 Ras expression 5-High JAK2 signaling inhibit the Bcl-xL deamidation MAPK pathway and the apoptotic response to DNA damage K STAT5 6- Could explain the progression of PMF to leukemia AKT MAPK P + STAT5 Homologous recombination + MDM2 Nucleus p53 Genetic instability p53 degradation

A low level of JAK2V617F would favour MPL signaling (JAK2V617F heterozygozity) G-CSFR Mpl ET EpoR Low kinase activity V617F N-term C-term SH2 FERM domain JH2 JH1 Cytokine receptor Pseudokinase Kinase domain Interacting domain domain

High level of JAK2V617F would permit strong EpoR signaling (JAK2V617F homozygozity) G-CSFR Mpl PV EpoR Intermediate kinase activity V617F N-term C-term SH2 FERM domain JH2 JH1 Cytokine receptor Pseudokinase Kinase domain Interacting domain domain

A high JAK2 signaling would induce a MK differentiation abnormality (JAK2V617F + ?) G-CSFR Mpl PMF Toxic effect EpoR High kinase activity V617F N-term C-term SH2 FERM domain JH2 JH1 Cytokine receptor Pseudokinase Kinase domain Interacting domain domain

Is it the only explanation for three disorders ? JAK2 V617F hits a hematopoietic stem cell as well as MPL W515L PV CD34 + PV CD34 + cells cells Human cells Human cells (majority of wt cells (majority of wt cells and minority of JAK2V617F cells ) and minority of JAK2V617F cells ) IV IV NOD-SCID NOD-SCID 1-4 months 1-4 months 36 19 56 39 3 69 100% 100% 80% 80% 60% 60% 40% 40% 20% 20% 0% 0% BFU-E EEC CFU-GM BFU-E EEC CFU-GM D0 W6

The proportion of JAK2V617F SRCs is different between PV and PMF patients Patients % JAK2V617F/ total % chimerism % JAK2V617F /total (colonies) % JAK2V617F/ total JAK2 JAK2 in granulocytes in CD45+c ells ( CD45+) PV1 69 15.6 0 (0/22) NA PV2 60 3.2 0 (0/56) NA PV3 60 4.0 NA NA PV4 44 7.8 4 (2/47) NA PV5 50 4.0 44 (4/9) NA Mean: Mean: 2.3% PV6 94 0.2 0 (0/1) 13% (23/172) 3 PV7 57 24.7 0 (0/2) 0 PV8 40 58.0 9 (3/34) 5 PV9 26 35.9 18 (14/79) 1 PMF1 50 43.9 100 (181/181) 67 PMF2 90 80.0 86 (60/70) 76 Mean: Mean: 58.2% PMF3 NA 0.8 NA 55 96% (248/259) PMF4 50 1.1 88 (7/8) NA PMF5 30 0.7 NA 46 PMF6 50 1.0 NA 47 PPVMF1 94 0.2 NA NA Mean: PPVMF2 NA 0.4 NA 95 95.5% PPVMF3 100 1.4 NA 96 PPVMF4 92 0.0 NA NA