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Mastocytosis Olivier Hermine MD,PhD CNRS UMR 8147 (Director M DY) Service d’hématologie Centre de référence des mastocytoses Hôpital Necker-Enfants Malades Faculté de Médecine Necker-Cochin Paris

Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

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Page 1: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Mastocytosis

Olivier Hermine MD,PhD

CNRS UMR 8147 (Director M DY)

Service d’hématologieCentre de référence des mastocytoses

Hôpital Necker-Enfants MaladesFaculté de Médecine Necker-Cochin

Paris

Page 2: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Mastocytosis

Olivier Hermine MD, PhD

Hematology Department, Necker Hospital

Centre National de référence des mastocytoses, Necker Hospital

CNRS UMR 81 47

Institut Imagine, Necker HospitalInstitut Imagine, Necker Hospital

Paris, France

Page 3: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Centre de Référence Des MastocytosesCEREMAST

Coordonnateur

Pr Olivier HERMINE

Co-coordonnateurs

Pr Christine BODEMER

Pr Olivier LORTHOLARY

Dr Stéphane BARETE pour EMSED

Laboratoires de référence

Pr Patrice DUBREUIL - INSERM

Pr Olivier HERMINE - CNRS UMR 8147Dr Sophie GEORGIN-LAVIALLE - CNRS UMR 81 47

Dr Sylvie FRAITAG -Anatomopathologie (peau)

Dr Danielle CANIONI - Anatomopathologie (moëlle)

Dr Vahid ASNAFI - Laboratoire d'hématologie

Responsible du projet CEREMAST

Dr Olivia CHANDESRIS

Attachée de recherche clinique

Isabelle HIRSCH

Psychologue

Daniéla MOURA

Secrétaire de coordination

Anne-Florence BELLAIS

Dr Vahid ASNAFI - Laboratoire d'hématologie

Pr Serge ROMANA - Cytologie

Anesthésiste Responsable

Dr Pascale DEWACHTER

[email protected]

Par téléphone : 01 40 61 54 11

Par fax : 01 40 61 56 02

CEREMAST - Faculté Necker,156, Rue de Vaugirard, 75015 Paris

Page 4: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Mast cells are tissue cells

They are prevalent in areaswhich interface directly with

the external environmentclose to blood vessels and

Mast cells are sentinentals with strategic location

close to blood vessels andnerve endings

They can respond veryrapidy to a stimulus

with the production of awhole array of mediators

from JS. Marshall, Nature Rev Immunol 420: 787- (200 4)

Page 5: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Mastocytosis : DefinitionMastocytosis : Definition

-- HETEROGENEOUS GROUP OF DISORDERS CHARACTERIZED BY HETEROGENEOUS GROUP OF DISORDERS CHARACTERIZED BY ABNORMAL GROWTH AND ACCUMULATION OF MAST CELLS IN ON E ABNORMAL GROWTH AND ACCUMULATION OF MAST CELLS IN ON E OR MORE ORGANS,OR MORE ORGANS,

-- CONSIDERED AS A MYELOPROLIFERATIVE DISORDER,CONSIDERED AS A MYELOPROLIFERATIVE DISORDER,-- CONSIDERED AS A MYELOPROLIFERATIVE DISORDER,CONSIDERED AS A MYELOPROLIFERATIVE DISORDER,

-- DIVIDED INTO CUTANEOUS FORMS AND SYSTEMIC FORMSDIVIDED INTO CUTANEOUS FORMS AND SYSTEMIC FORMS

-- CLASSICAL SPONTANEOUS RESOLUTION FOR PEDIATRIC CA SESCLASSICAL SPONTANEOUS RESOLUTION FOR PEDIATRIC CASE S

-- ASSOCIATION WITH HEMATOLOGICAL DISORDERSASSOCIATION WITH HEMATOLOGICAL DISORDERS

Page 6: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•I - Localized•Cutaneous mastocytosis

•Mast cell sarcoma

•Extracutaneous mastocytoma

••CONSENSUS CLASSIFICATION OF MASTOCYTOSIS CONSENSUS CLASSIFICATION OF MASTOCYTOSIS ••(simplified From Valent et al, 2001)(simplified From Valent et al, 2001)

•Extracutaneous mastocytoma

•II - Generalized•Systemic mastocytosis

•- Indolent (no organ dysfunction)

•- With an AHNMD

•- Agressive (organ dysfunction)

•Mast cell leukemia (> 20%MC in BM)

Page 7: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

PhysiopathologyPhysiopathology

Page 8: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

MCTC MCT

SCF

����CD 34+c-kit high

MCC

IL-4SCF

?

TISSUES

IL-4Survival :SCFNGFIL-4IFN- γγγγ…...

Stem Cell CD34+Bone marrow

CIRCULATIONSCFIL-10IL-6

c-kit highFcεεεεRI negCD13+

SimplifiedSimplified pathwayspathways of of humanhuman MC MC differentiationdifferentiation

Page 9: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•• CC--kit activation pathwaykit activation pathway•• CC--kit activation pathwaykit activation pathway

•Lyn•Lyn

Page 10: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Mutations de c-kit dans les lignées cellulaires et chez les patients atteints de mastocytose

P815 souris D814Y PTFMA3 souris d573-579 JMRBL-2H3 rat D817Y PTHMC-1 humain V560G JM

D816V PT

Lignées cellulaires

ENFANT ADULTE CAS FAMILIAUX

Pas de mutationou E839K

formes extensives:D816VD816YD816F

Pas de MutationD816V/H-Systémique (80%)-Atteinte cutanée (30-50%)

V560G (rare)D820G (leukemia)

Patients avec Mastocytose

Page 11: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Mastocytosis and c-Kit mutations

•Cutaneous biopsies -> RT-PCR

•Cutaneous mastocytosis UP and systemic mastocytosis

•P. Dubreuil, F Palmérini , L Nasca , (IPC Marseille - réseau AFIRMM – M Arock, O Hermine )

Page 12: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Augmentation de la sensibilité de détection de la mutation D816V

PCR: 2295S / 2661R

(exons 17 to 18 Fragment of 366 pb)

Ig1 Ig2 Ig3 Ig4 Ig5

TM JM TK1 TK2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

WT D816V

Digestion BsmaI

Nested-PCR amplification

Sequence

Page 13: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•D479N

•V560G� ∆∆∆∆418

� ∆∆∆∆564-576

•E860G� ∆∆∆∆419 (12)

•InsFF419

•C443Y•S476I

•ITD501-502(2)

•D572A•D816Y(4)

•D816I•D816V(353)

•Ig1 •Ig2 •Ig3 •Ig4 •Ig5

•TM •JM •TK1 •TK2

•1 •2 •3 •4 •5 •6 •7 •8 •9 •10 •11 •12 •13 •14 •15 •16 •17 •18 •19 •20 •21

� ∆∆∆∆417-418 D419Y

•n=603•Human mastocytosis

•Collaboration : K Hahn Houston USA

•MCL leukemia

•And Sarcoma•Systemic•Pediatric

•Ig1 •Ig2 •Ig3 •Ig4 •Ig5

•TM •JM •TK1 •TK2

•1 •2 •3 •4 •5 •6 •7 •8 •9 •10 •11 •12 •13 •14 •15 •16 •17 •18 •19 •20 •21

•Dog mast cell tumor� ∆∆∆∆826826826826−−−−828- InsDT•Q430R

•ins421 aa45

•S479I4

•N508I

•Del/Ins555-559(5)

•ITD571-590(15)

•n=88

•Team 4 : Cell Signaling, Hematopoiesis and Mechanisms of Oncogenesis

•ITD

•ITD505-508

•K509I(6)

•ITD502-503(4)

•30% of the dogs are mutated

•And Sarcoma•Systemic

•Mastocytosis 85%

•Indolent and agressive

•Pediatric

•Mastocytosis 85%

Page 14: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Adultes : STAT1_3_5 • Enfants : AKT – GSK3b

•Mutations ECD and D816X signalisation

•P. Dubreuil

Page 15: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Differential colony-forming ability of ECD and D816X mutants expressing Rat2 cells

•Without

•SCF

•With

•SCF•SCF

Page 16: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Outcome and mutations of c-kit

Patients mastocytosisStatut of c-Kit

WTMutation autre que

D816VD816V

Children (N= 59) 15% 46% 39%

Adults (N= 485) 28% 3% 69%

•Mutation %

•age

•Mutation %

•persistent

•Mutations

•ex8 & ex9

•puberty

•Mutations

•D816V

•regressive

•50

•75

•25

Page 17: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Targeting telomerase and telomeres

•AZT

•P53•P53

•Senescence

Page 18: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Conséquences du raccourcissement des télomères•Catalado RT, NEJM 2009

•Crise

•Instabilité

télomérique

•Sénescence

•Télomères : taille

critique

•Cellule somatique

•p53

•pRb •Cellule

immortalisée

•Stabilisation des télomères

•Divisions cellulaires

•Taille des

télomères

•Cellule souche

•Cellule souche

Page 19: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Re

lati

ve t

elo

me

rase

act

ivit

y

•In Culture Mast cells dye by senescence (6 weeks)

•Telomerase activity ?

•MC

•WT

•MC

•D816V

•R

ela

tive

te

lom

era

se a

ctiv

ity

Page 20: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Figure 1 : « differential expression of telomerase and telomerase activity in children and adults

mastocytosis »•B •C •D

•E •F

•A

•Children

•N=15

•Adults

•N=16

•%

hT

ER

T+

c-K

it+

ce

lls

•G

•h

TE

RT

/c-K

it m

RN

A e

xpre

ssio

n

•Children

•n=60

•Adults

•n=48

•E

•MC

•WT

•MC

•D816V

•R

ela

tive

te

lom

era

se a

ctiv

ity

•F •G

•Children n=46

•n=17•n=18 •n=11

•p = 0,043

Page 21: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Fibroblastes primaires humains D816V

•DAPI •Cy3 •Merge

•Mesure de la longueur des télomères

•Par analyse FISH Cy3-(CCCTAA)3

•Fibroblastes primaires humains Del417-419 InsY

•DAPI •Cy3 •Merge

Page 22: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•In vitro studies in transfected fibroblasts and BMMC

•A •B

•***•*

•**

•WT •Empty •Del417/ •AY502 •D816V

•*•***

•WT •Empty •Del417/ •AY502 •D816V

•C

•WT •Empty

Vector

•Del417/

8+D419Y

•AY502

/503•D816V•WT •Empty

Vector

•Del417/

8+D419Y

•AY502

/503•D816V

•WT •EV •417 •816•502 •EV •417 •816•502

•D •E

Page 23: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Mutation Val560Gly

•(JM)

•C-Kit-hTERT

•A: moelle osseuse du patient

•10 Kb

•7,4 Kb

•M L. rate

•CT sain, apparié

en âge

•TRF2

•PML

•DAPI

•Colocalisation

PML et TRF2

•Cytospin sarcome

•A: moelle osseuse du patient

•B: sarcome du patient

•D: témoins (ISM)

•C: témoin (ISM)

•Collaboration Cytogénétique Serge Romana, G Soler

•Collaboration Curie •Arturo Londono

Page 24: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Role of Telomerase activity

•Mutation

•D816V

•(Exon 17)

•C-Kit

• Mastocyte Survival

•=> immortalisation

•Indolent Mastocytosis

•Télomerase activity

•Spontaneous

Regression

•Mutation

•Exons 8 à 11

•C-Kit et WT•Proliferation

•Senescence

•-No telomerase

activity

•Short telomeres

•SCF

Regression

•Tumor progression

- Instabilité génétique

- ALT

Page 25: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Heterogenity of diseases associated

with D816V mutations

Agressive and Hematological diseases Agressive and Hematological diseases

association vs Indolent

Page 26: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•5-me-Cytosine •5-hme-Cytosine

•Tet2

•Tet2 is involved in the conversion of 5 meC to 5hmeC

•at specific genomic loci

•Cytosine

•Dnmt•Inhibitors

•(e.g. RG108)

Page 27: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•5-me-Cytosine •5-hme-Cytosine

•Tet2

•Tet2 is involved in the conversion of 5 meC to 5hmeC

•at specific genomic loci

•Cytosine

•Dnmt•Inhibitors

•(e.g. RG108) •Mutations in SMP and AML

•CMML++

•CMML is associated with MS

Page 28: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Tet2 mutations are frequent and are correlated to aggressive disease

•Mastocytosis Patient Cohort:

Page 29: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•KitD816V increases proliferation of Tet2-deficient BMMCs

Page 30: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

0 3 6 10 12 14

•Li

ve C

ell C

ou

nts

(lo

g 10)

•(-/-)

•n=4

•(+/-)

•n=4

•(+/+)

•n=4

•8

•0

•2

•4

•6

•7

•1

•3

•5

•A •B

•The combination of Tet2 loss and c-Kit activation provides factor independent survival of

BMMCs

•Time after IL3 withdrawal (Days)

•0

•C

•Gating on

•GFP +

Page 31: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Bone marrow derived Tet2-deficient mast cells infected with c-KitD816V have a competitive

growth advantage

CD117

•cKitD816V+ •cKitD816V+ •cKitD816V+

� Confirmed in two independent Tet2-KO models

(lacz and flox)

� Consistent whether infections are done using

« young » or « old » MC cultures (old > 80 days

culture with IL3)

•CD117

•Tet2(+/+)

•GFP

•Tet2(+/-) •Tet2(-/-)

Page 32: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•TET2 MUTATION

•Model of Oncogenic Co-operation

••KITD816V KITD816V

••OTHER MUTATIONSOTHER MUTATIONS••HETEROGENEOUS DISEASE PHENOTYPEHETEROGENEOUS DISEASE PHENOTYPE••AGRESSIVE DISEASEAGRESSIVE DISEASE

•Proliferation

•Differentiation

••HETEROGENEOUS DISEASE PHENOTYPEHETEROGENEOUS DISEASE PHENOTYPE••AGRESSIVE DISEASEAGRESSIVE DISEASE

Page 33: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Indolent

•Mast cell tumor

•Htert activiy

•C-kit D816V

•Survival

•Immortalization

•Mast cell tumor

• regression•C-kit (WT exon 8,9,11)

•Proliferation

•SCF

•Survival

•No immortalization

•No htert activity

•Telomeres shortening•Mast cell

•progressiopn

•Genetic instability

•Htert expression

Page 34: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

AHNMD• Fréquence 30%• Physiopathologie?• Pronostic habituellement mauvais

• Hémopathie myéloïde : SMP, SMD, SMP/SMD– LMMC: précurseur commun?, monocytes D816V, Mutation TET2 30%, évolution

possible vers LAM– LAM : t(8;21) ++, présence SM souvent occulte et péjore le pronostic de la LAM. De

même, présence LAM péjore pronostic de la MS– LMC: pas de BCR-Abl– LMC: pas de BCR-Abl– Hyper-éosinophilie (<20%):

– SHE, leucémie chronique à éosinophiles– FIP1-PDGFRa = imatinib– SM-eo: pas de retentissement habituel cardiaque ou pulmonaire

• Hémopathie lymphoïde: rare. NHL, gammapathie mc

• Si indiqué, traitement dissocié de l’une et l’autre pathologie et traiter l’hémopathie comme si elle était de novo.

• Pas d’effet des HD-CT sur la mastocytose

Page 35: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Mastocytosis Heterogeneity and c-kit

•Tet2

•ASXL1

•CMML

•C-kit 816 V

•negative

•C-kit 816 V

•Other ckit mutations •Other ckit mutations

•other ?

•NHL

•Cytokines (CD40L)

•Tet2

•ASXL1

•C-kit 816 V

•Tet2

•MDS

•CMML

•AML

•Sarcoma

•MCL

•Indolent Masotcytosis

•Agressive mastocytosis

•C-kit 816 V •JAK2,

• FIP1L1-PDGFR,

•Regression

•C-kit 816 V

•Other ?•Pediatric forms

•Other ckit mutations

•WT c-kit

•Adult Forms

•Sarcoma

•MCL

•Other ckit mutations

•WT c-kit

•Others SMP

•Agressive

•Masotcytosis (rare)

•Tet2

•ASXL1

•others

Page 36: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Effect of kinase Inhibitors on Mast cellsand Mastocytosis

C-kit inhibitionC-kit inhibition

STI 571 ?

Page 37: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Effect of STI 571on proliferation of cell lines with juxtamembrane or V814 c-kit mutations

80

100

120

% C

PM

0

20

40

60

0 0.1 1 10

µM

% C

PM

Ba/F3 Kit+IL3

Ba/F3 Kit+SCF

∂27

FMA 3

KIT G559

IC2 V814

P815 (V814)

Page 38: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

FMA3 K∆27 KG559 KV814 P815

STI571 (µM) 0 1 10 0 1 10 0 1 10 0 1 10 0 1 10

KWT

.1 1 10

KL - + + - - - - - - - - - - - - - - -

IP anti KIT / Blot anti PY

000

- ++

--

175 kDa

83 kDa

MW

Ct

KIT

Effect of STI 571on wild type, juxtamembrane or V81 4c-kit phosphorylation

IP anti KIT / Blot anti PY

Total cell lysate & Blot anti Grb2

IP anti KIT / Blot anti KIT

--

175 kDa

83 kDa

MW

KIT

Grb226 kDa -

Page 39: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Effects of STI571 on Mastocytosis

• No effect on Mast cells with C-kit mutations within the catalytic domain (D816V)

• In vitro effect on Mast cells without catalytic domain C-kit mutations

Page 40: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

c-kit Mutations in Mastocytosis

NH2

cc

cc

cc

Akin et al. Blood. 103:3222, 2004

•Location: Exon 10 (Transmembrane)•Type: Germline•Clinical features:

Childhood onset CMSSM in early 20sWell-differentiated phenotype

COOH

cc

F522C

Well-differentiated phenotype•Functional studies: Gain-of-function•Inhibition by imatinib: Yes (in vitro and clinically)

Page 41: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Therapy with imatinib in a patient with transmembrane c-kit mutation: Proof of concept

150

200

Try

ptas

e (n

g/m

l)

100 mg/d

200 mg/d

300 mg/d

0 25 50 75 100 1250

50

100

Days

Try

ptas

e (n

g/m

l)

300 mg/d

400 mg/d

Akin, Fumo, Yavuz, Lipsky, Neckers, Metcalfe. Blood, 103:3222, 2004

Page 42: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR
Page 43: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

PKC412 (N-benzyl staurosporine)

• Inhibits PKC, VEGFR, Kit, PDGFR, flt3

• Phase I/II trials in hematologic and solid hematologic and solid tumors– Nausea, vomiting,

diarrhea, fatigue

• Inhibits D816V c-kit•Gotlib et al. Blood, in press, 2005

•BAF3 D816V cells

Page 44: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

PKC 412

• Agressive mastocytosis

• Mastocytosis associated with C-kit D816V mutation

Page 45: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Signaling of PI-3k/Akt/mTOR•C-KIT

•-•PI •3 kinase

•AKT

•-•m•TOR •RAPAMYCINE

•1•Cyclin D•CDK’s

•G•1 •S

•Cell division

•G•0

•(resting cell)

•+

•P-p70S6k

•Translation/apoptosis

•4EBP/eIF4E

Page 46: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•BMMC

•Lignée BMMC•cellules mastocytaires dérivées de moelle osseuse de souris

•transgéniques pour le c-kit humain

•BMMC

•c-kit wt•BMMC

•c-kit 816

•La mutation D816V du c-kit confère à la cellule BMMC la sensibilité à la Rapamycine

Page 47: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Rapamycin and MS

•Stop

IFN

•CDA

1

•CDA

2

•Start

IFN

•Start Rapamycin

Page 48: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR
Page 49: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Masitinib formula: C28H30N6OS

•Molecular weight:

•AB1010 (mesylate salt): 594.76

•Masivet® belongs to the ATP binding pocket molecules kinaseinhibitor 2-amino-4-aryl-thiazole family

•c-Kit •c-Kit

•Proliferation

•AB1010

•Proliferation

Me

N

S

NH

NH

O

N

N

N

Me

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1010,10,050,01

DO

490

nm

AB 1010 (µM)

Inhibition of c-Kit phosphorylation

•AB1010 (mesylate salt): 594.76

•AB1003 (free base): 498.66

•Proliferation •Proliferation

•AB1010

•Anti-c-Kit

•Anti-Phospho Kit Tyr 729

•Anti-Phospho Tyr 4G10

Page 50: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Masitinib formula: C28H30N6OS

•Molecular weight:

•AB1010 (mesylate salt): 594.76

•Masivet® belongs to the ATP binding pocket molecules kinaseinhibitor 2-amino-4-aryl-thiazole family

•c-Kit •c-Kit

•Proliferation

•AB1010

•Proliferation

Me

N

S

NH

NH

O

N

N

N

Me

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1010,10,050,01

DO

490

nm

AB 1010 (µM)

Inhibition of c-Kit phosphorylation

•AB1010 (mesylate salt): 594.76

•AB1003 (free base): 498.66

•Proliferation •Proliferation

•AB1010

•Anti-c-Kit

•Anti-Phospho Kit Tyr 729

•Anti-Phospho Tyr 4G10

Page 51: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Masivet® potently and selectively inhibits c-kit, PDGF receptors, and LynB

••Major target of Major target of MasivetMasivet® (IC® (IC5050 below 1µM)below 1µM)Major targets

(IC50 in nM)

Inhibition of cell proliferation Inhibition of kinase activity

ligandTransfected cells Tumor cell lines

Human c-Kit

WT 150 150 (TF-1) 200 SCF

EC (exon 9) 100 NA NA -

JM (exon 11) 3 50 (HMCI-a155) NA -

TK1 (exon 13) ND 40 (GIST 882) ND -

TK2 (exon 17) 5000 5000 (HMCI) > 10,000 -

Dog c -Kit

Masivet® is a novel, orally administered, protein-t yrosine kinase inhibitor, which potently and selectively inhibits c-kit, PDGF recep tors, and Lyn.

Dog c -Kitd Kit WT 100 NA ND SCF

d Kit EC exon 8 20 NA NA -

d Kit EC exon 9 100 NA NA -

d Kit JM exon 11 5 20 (C2) NA -

Murine c-Kitm Kit WT 150 200 (BMMC) ND SCF

m Kit JM exon11 5 NA NA -

Platelet-derived growth factor (PDGF) receptors

PDGFRα WT 300 - 540 PDGF BB

FIP1L1-PDGFRα - 0.25 (EOL-1) ND -

PDGFRβ 30 ND 800 EGFR-chimera + EGF

Intracellular kinases

LynB NA NA 510 -

Page 52: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Masitinib inhibits Mast cell degranulation and migration

Page 53: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

••Human : A model for DogHuman : A model for Dog

••and “vice versa”and “vice versa”

•53

Page 54: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Mast Cell Tumors in Dogs

• Most common cutaneous tumor– 16-21% of all canine

cutaneous tumorscutaneous tumors

• Tissue mast cells in the dermis

• Poor survival <6 months in grade III tumors

Page 55: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

D479N

V560G∆∆∆∆418

∆∆∆∆564-576

E860G∆∆∆∆419 (12)

InsFF419

C443YS476I

ITD501-502(2)

D572AD816Y(4)

D816ID816V(353)

Ig1 Ig2 Ig3 Ig4 Ig5

TM JM TK1 TK2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

∆∆∆∆417-418 D419Y

n=603Human mastocytosis

Collaboration : K Hahn Houston USA

Can we compare mastocytosis with dog mast cell tumors ?

Ig1 Ig2 Ig3 Ig4 Ig5

TM JM TK1 TK2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Dog mast cell tumor∆∆∆∆826826826826−−−−828- InsDTQ430R

ins 421 aa45

S479I4

N508I

Del/Ins 555-559(5)

ITD571-590(15)

n=88

Team 4 : Cell Signaling, Hematopoiesis and Mechanisms of Oncogenesis

ITD

ITD505-508

K509I(6)

ITD502-503(4)

30% of the dogs are mutated

Page 56: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Case Report in Mast Cell Tumor.•Case Report in Mast Cell Tumor.

•DAY 0 •DAY 3 •DAY 18

Page 57: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

•Case Report in Mast Cell Tumor.•Case Report in Mast Cell Tumor.

•Day •-•40• •Day 0 • •Day 15 • •Day 40••(treatment start) •

Page 58: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

0. 25

0. 50

0. 75

1. 00

•EFFICACY of KINAVET on MAST CELL TUMOUR•Pivotal phase III study (n=202)

•Time To Progression – FIRST LINE

ITT population Median TTP (days)

Category n Mb Plc P

All First Line (FL) 85 253 75 0.001

Mutated c-Kit 25 NR 83 0.009

Wild-type c-Kit 53 253 66 0.008

•All First Line

0. 00

0. 25

0. 50

0. 75

1. 00

Ti me t o progressi on (days)

0 100 200 300 400 500 600

STRATA: group=AB1010 Censored group=AB1010 group=Pl acebo

0. 00

0. 25

0. 50

0. 75

1. 00

Ti me t o progressi on (days)

0 100 200 300 400 500

STRATA: group=AB1010 Censored group=AB1010group=Pl acebo Censored group=Pl acebo

0. 00

Ti me t o progressi on (days)

0 100 200 300 400 500 600

STRATA: group=AB1010 Censored group=AB1010group=Pl acebo Censored group=Pl acebo

Wild-type c-Kit 53 253 66 0.008

•Mutated c-kit •Wild-type c-kit

•Mb, masitinib; Plc, placebo; NR, not reached

Page 59: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Case Report of Agressive MC

• Female 67y past history of Flushes, Diarrhea, abdominal pain, syncopes.

– Hb 10,6, Plt 124000, wbc 8100; 4600 PNN, 7 % circulating mastocytosis

– Bone marrow aspiration 77% pathologic mast cells – Bone marrow aspiration 77% pathologic mast cells

– Bone marrow biopsy : 90 % infiltration

– Tryptase sérique 351 µg/L (N<15)

– Histamine sérique 14314 nmol/L (N<15)

– Caryotype: normal (46XX)

– Flow cytometry: 40% Ckit+, 12% CD25+, 2%

Page 60: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

Biological findings

•Deletion 501-502

•Blood •Bone Marrow

Page 61: Olivier Hermine MD,PhD - … · Mastocytosis Olivier Hermine MD, PhD HematologyDepartment, Necker Hospital Centre National de référence des mastocytoses, Necker Hospital CNRS UMR

In vitro effect of MasitinibPostive C

ontorol

Cos t

ransfected

Cos t

ransfected +masitinib

Cos t

ransfected+masitinib

1

•Cos

•Postive C

ontorol

•Cos t

ransfected

•Cos t

ransfected +masitinib

0.1

•Cos t

ransfected+masitinib

1

•Deletion 501-502