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 Essential Thrombocythemia (ET) CML Classical MPNs

Rare and unclassified MPNs

Polycythemia vera (PV) Myelofibrosis (PMF) Bcr-abl

PDGFRa FGFR1 Kit

B cell B cell PEo PEo PB PB Mono Mono PN PN CFU-MK CFU-MK CFU-baso CFU-basoPre-B Pre-B Pre-T Pre-T CFU-Eo CFU-Eo CFU-GM CFU-GM BFU-E BFU-E CFU-E CFU-E RBC RBC

PRECURSORS PRECURSORS

Mature cells Mature cells

Macrophage Macrophage PLT PLT

HSC HSC

T cell T cell DC DC NK NK

PROGENITORS PROGENITORS

Normal hematopoiesis

BFU-E BFU-E CFU-E CFU-E B cell B cell PMEo PMEo PMB PMB Mono Mono PMN PMN CFU-MK CFU-MK Pre-B Pre-B Pre-T Pre-T RBC RBC Macrophage Macrophage PLT PLT T cell T cell DC DC NK NK CFU-GM CFU-GM CFU-Eo CFU-Eo CFU-baso CFU-baso

MPNs MPNs are clonal diseases involving the HSC leading to increase blood cell production

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 450x109/L – Megakaryocyte hyperplasia with increased number of large megakaryocytes – No criteria of other MPN

Molecular events and classical MPNs

PV

?

ET PMF

JAK2V617F

95%

JAK2V617F JAK2V617F MPLW515

JAK2 exon 12 JAK2 exon 12

3% 3%

50% 50% 50% 50%

8% 8%

1% 1% MPL W515 MPL W515 LNK (exon 2) LNK (exon 2) 1% 1%

8% 8%

LNK (exon 2) LNK (exon 2) MPL W515 MPL W515 c-CBL c-CBL 6% 6%

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

LNK exon 2

Oh, Blood 2010

CBL

Grand, Blood 2010

PV is a homogeneous disorder related to a JAK2 constitutive activation requiring cytokine receptors

JAK2 fusion:T-cell lymphoma, Adelaide et al

JH1 JH2 JH4-JH3 kinase pseudokinase SH2 FERM JH7-JH5 TEL TEL TEL

JAK2 JAK2 fusion: CML, Peeters et al JAK2 fusion: ALL, Peeters et al JAK2 fusion: ALL, Lacronique et al

PCM1 PCM1 PCM1 PCM1

JAK2 fusion: CML, Bousquet et al JAK2 fusion: CML/ALL, Reiter et al JAK2 fusion: MPD, Murati et al

kinase pseudokinase SH2 FERM kinase pseudokinase SH2 FERM

V617F K539L

Homodimeric cytokine receptors: EpoR, MPL, GCSFR GHR, PrlR

Cytokine receptors sharing βc subunit: IL-3R, IL-5R, GM-CSFR Cytokine receptors sharing gp130 subunit: IL-6R, IL-11R, OSMR, LIFR, Cytokine receptors sharing γc subunit: IL-2R, IL4R IL-7R, IL-9R, IL-15R, IL21RInterferons & other Type II receptors: IFNα, IFNβ, IFNγ, IL-10R, IL-19R, IL- 20R, IL-22R, IL- 24R, IL28R, IL29R JAK2 JAK2 JAK1/JAK2, TYK2 JAK1, JAK2/ JAK3 JAK1, JAK2 and TYK2 (IFNs) Type I Type II

Cytokine receptors and JAK2

Requirement of cytokine receptors

V617F

C-term N-term JH1 JH2 Kinase domain Pseudokinase domain Cytokine receptor Interacting domain FERM domain SH2

Y114A

Wernig et al. Blood 2008

ET is a heterogeneous disorder related to a MPL constitutive activation

JAK 2 JAK 2 PI-3K C-Akt P STAT3/ 5 STAT3 STAT3 P P

Nucleus

P P P P P P P P ER K Grb 2 Sos Ras Raf ME K Shc STAT 3 P Y Y Y Y Y Y IRS2 PKCζ

JAK2/STAT essential for proliferation

Constitutive activation

• f MPL

EpoR

pairs of cysteines WSXWS box1 box2

MPL

JAK2V617F JAK2V617F Constitutive activation

• f MPL and EPOR

JAK2V617F

WT TMPL WT MPL + Tpo ∆5 MPL MPL W515A/L/K

T p

• Motif RWQFP

S505N

T487A

T487A S505N

MPL mutants LNK mutants

ET is a heterogeneous clinical and biological disorder

AL AML4 ET ET ET>PV>MF>AL ET ET AML ET>MF>AL ET

Group 1 Group 2 Group 3

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

• 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

Normal spleen Splenomegaly Giant platelets Tear drop erythrocytes Erythro-myelemia Hyperleukocytosis with neutrophil polymorphs Femurs Myelofibrosis Osteosclerosis

Fibroblast proliferation

 OPG

MARROW FIBROSIS OSTEOSCLEROSIS Osteoblast proliferation

 TGF-β1

Local activation

Decrease of mature

• steoclasts

Implication of monocyte Stromal cell stimulation Implication of monocyte

 IL-1α NF-κB

TPO JAK2V617F

Mpl 515 MEGAKARYOCYTE

TF TPO JAK2 J A K 2 BRAF MEK1/2 ERK1/2 EGR1 p21 PI3K AKT STATs RAF1

Inflammatory transcriptome

IL6 IL8 IL1 TGFβ IL6 IL8 IL1 TGFβ

Inflammatory secretome

IL10 IL10

Cell cycle arrest DNA damage

Cellular senescence

ROS DNA Repair

High MPL activation normally induces senescence

In normal MK differentiation, proliferation is limited by a senescence process

pERK p21 Actin D0 D3 D6 D9 Normal Control PMF p21 Actin

PMF Healthy donor p < 0.03

p21 expression (2-∆Ct (p21-HPRT)) 0.01 0.1 1 10 0.1 1 10 100

PMF Healthy donor p < 10-2

SA-ß-Gal-positive cells (%)

This senescence process is inhibited in PMF (Requirement for several genetic or epigenetic abnormalities ?)

High JAK2 signaling induces genetic instability and thus disease progression

Cytoplasm Nucleus

+

Homologous recombination

Genetic instability

+

P P P P P Y Y Y Y Y Y

EoR

P JAK2

JAK2

V617F V617F

p53

MDM2

p53 degradation

P STAT5 S O C S

PI-3K

Grb 2 Sos

Ras Ras AKT MAPK K MAPK

Shp-2 Shp-1 C I S STAT5 STAT5

1- High JAK2 signaling inhibits p53 function 2- High JAK2 signaling stimulates HR 3- High JAK2 signaling induces genetic instability 6- Could explain the progression of PMF to leukemia 4- High JAK2 signaling induces H3Y41 and LMO2 expression 5-High JAK2 signaling inhibit the Bcl-xL deamidation pathway and the apoptotic response to DNA damage

JH1 JH2 Kinase domain Pseudokinase domain Cytokine receptor Interacting domain

V617F

C-term N-term FERM domain SH2

Low kinase activity

ET

Mpl

EpoR

G-CSFR

A low level of JAK2V617F would favour MPL signaling (JAK2V617F heterozygozity)

PV

JH1 JH2 Kinase domain Pseudokinase domain Cytokine receptor Interacting domain

V617F

C-term N-term FERM domain SH2

Intermediate kinase activity

Mpl

EpoR

G-CSFR High level of JAK2V617F would permit strong EpoR signaling (JAK2V617F homozygozity)

JH1 JH2 Kinase domain Pseudokinase domain Cytokine receptor Interacting domain

V617F

C-term N-term FERM domain SH2

High kinase activity

PMF

Toxic effect

Mpl EpoR G-CSFR

A high JAK2 signaling would induce a MK differentiation abnormality (JAK2V617F + ?)

Is it the only explanation for three disorders ?

JAK2 V617F hits a hematopoietic stem cell as well as MPL W515L

Human cells Human cells

(majority of wt cells (majority of wt cells and minority of JAK2V617F cells ) and minority of JAK2V617F cells )

PV CD34 PV CD34+

+

cells cells NOD-SCID NOD-SCID

1-4 months 1-4 months

IV IV

0% 20% 40% 60% 80% 100% BFU-E EEC CFU-GM 0% 20% 40% 60% 80% 100% BFU-E EEC CFU-GM

D0 W6 36 19 56 39 3 69

Patients % JAK2V617F/ total JAK2 in granulocytes % chimerism ( CD45+) % JAK2V617F /total (colonies) % JAK2V617F/ total JAK2 in CD45+c ells

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 PV6 94 0.2 0 (0/1) 3 PV7 57 24.7 0 (0/2) 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 PMF3 NA 0.8 NA 55 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 PPVMF2 NA 0.4 NA 95 PPVMF3 100 1.4 NA 96 PPVMF4 92 0.0 NA NA

Mean: 13% (23/172) Mean: 96% (248/259) Mean: 2.3% Mean: 58.2%

The proportion of JAK2V617F SRCs is different between PV and PMF patients

Mean: 95.5%

Clonal amplification TET2 mutations Is JAK2 signaling sufficient to induce a clonal dominance at the level of HSC ?

(Requirement for other mutations such as TET2 ? )

TET2 defects (including substitutions of residues

• utside the

conserved domains) Total frequency TET2 defect (without substitutions

• utside the

conserved domains) Total frequency PMF JAK2 VF 6 31 0,19 6 31 0,19 PMF JAK2/MPL neg 5 25 0,20 5 25 0,20 PMF MPL 515 1 3 0,33 1 3 0,33 PV JAK2 ex12 1 0,00 1 0,00 PV JAK2 VF 27 170 0,16 23 170 0,14 ET JAK2 VF 19 172 0,11 17 172 0,10 ET JAK2/MPL neg 10 108 0,09 5 108 0,05 ET MPL 515 1 9 0,11 1 9 0,11 Post MPD MF 1 10 0,10 1 10 0,10 total 70 529 0,13 59 529 0,11 JAK2 VF 52 373 0,14 46 373 0,12 MPL 515 2 12 0,17 2 12 0,17 JAK2 / MPL Neg 15 134 0,11 10 134 0,07

Commited Progenitors (BFU-E, CFU-GM) Precursor cells Erythroblasts Granulocytes B cells, NK Lympho- Myeloid progenitors CD34+/38- Thrombocytosis Erythrocytosis Erythrocytosis Homo. JAK2V617F Normal marrow PV homozygous PV heterozygous ET (heterozygous) PMF Fibrosis

Conclusion

• PV is a homogeneous disease related to an activation of

JAK2 through myeloid cytokine receptors

• ET is a more heterogeneous disorder presently related to a

moderate MPL activation

• PMF is a even more heterogeneous disorder requiring an

high MPL activation associated with other genetic events. Other mechanisms might be involved.

Acknowledgments

INSERM U985 Villejuif Olivier Bernard Véronique Della Valle Roland Berger H St Antoine, H la Pitié Paris Christine Bellane Albert Najman Cécile Saint Martin Ludwig Institute for Cancer Research (Brussels) Stefan Constantinescu Christian Pecquet Judith Staerk INSERM U1009 Villejuif Eric Solary François Delhommeau Chloe James Sabrina Dupont Nicole Casadevall Stéphane Giraudier Ronan Chaligne Rodolphe Besancenot Jean-Pierre Le Couédic Isabelle Plo Jean Luc Villeval Catherine Lacout Caroline Marty Mayouka Nakatake Siham Boukour