5a. Tyrosine kinases

TIROSIN-CHINASI

Recettori transmembrana per fattori di crescita

Enzimi citoplasmatici

CITOPLASMA

NUCLEO

ABL1

JAK2 Tirosin-chinasi citoplasmatica

PDGFRBPDGFRAFGFR1C-KITMPLEGFRRecettori(Tirosin Chinasi)

TIROSIN-CHINASI

RECEPTOR FAMILY

Tyr Kinase domains

Cell Membrane

Extra Cellular

Intra Cellular

Phosphorilation - Dimerisation - Signal Transduction ( RAS; STAT; MAPK…)

JAK 2MPL

ARG (ABL2) FLT3KIT

PDGFRαPDGFRβFGFR1

DISORDINI MIELOPROLIFERATIVI

• LEUCEMIA MIELOIDE CRONICA: Ph-positiva

Ph-negativa, BCR-ABL 1 positiva

•

• Ph-negativa

Myeloproliferative Disorders

CMLBCR/ABL1

Others

PV

MFET

Trombocitemia essenziale

Mielofibrosi idiopatica

Policitemia vera

CITOPLASMA

NUCLEO

ABL1

JAK2 Tirosin-chinasi citoplasmatica

PDGFRBPDGFRAFGFR1C-KITMPLEGFRRecettori(Tirosin Chinasi)

TIROSIN-CHINASI

BCR/ABL1

ABL1/9q34BCR/22q11

Ph

t(9;22)(q34;q11)

CITOPLASMA

NUCLEO

Apoptosi

Proliferazione

BCR ABL1 Adesione Cellulare Tirosin Chinasi

TERAPIE MOLECOLARI(MIRATE/BERSAGLIO/TARGET/INTELLIGENTI)

TERAPIE MOLECOLARI(MIRATE/BERSAGLIO/TARGET/INTELLIGENTI)

Glivec (imatinib mesilato) Inibitore selettivo della tirosina-chinasi (Bcr-

Abl)Meccanismo d’azione

Goldman JM, Milo JV, NEJM 2001, 344:1084-1086

ProteinaBcr-Abl

Substrato

Substrato

Effettore

ATP

P

Y

Y

PP

P

ProteinaBcr-Abl

Substrato

Substrato

Effettore

GLIVEC

Y

Y

Y = Tirosina

P = Fosfato

Inibitori Tirosin chinasi

• Imatinib

• Nilotinib

• Dasatinib

• Sumitinib

• …………

• …………

% risposta

0

100

80

2030

50

Mesi dall’inizio del trattamento

90

7060

40

10

0

GLIVEC

IFN- + Ara-C

83%*

20%

* p<0,001123 96

% risposta

0

100

80

2030

50

Mesi dall’inizio del trattamento

90

7060

40

10123 96 1815 210

GLIVEC

IFN- + Ara-C

96%*67%

Risposte citogenetiche

Risposta citogenetica completa 0% Ph+

Risposta citogenetica parziale 1-35% Ph+

Studio IRISProbabilità di risposta citogenetica maggiore

(stima a 12 mesi)

Studio IRISProbabilità di risposta ematologica completa

(stima a 12 mesi)

Risposta ematologica completa

Leucociti < 10 x 109/LPiastrine < 450 x 109/LAssenza di blasti, basofili < 20%Assenza di malattia extramidollare

INIBITORI TIROSIN-KINASI

CGP 571 48 (Glyvec, STI571)

appartiene alla classe dei composti 2-

fenilaminopirimidine

prodotti sulla base della struttura del sito di

legame dell’ATP per le protein-kinasi

CMLBCR/ABL1

Others

PV

MFET

Myeloproliferative Disorders

JAK2 MPL

CITOPLASMA

NUCLEO

ABL1

JAK2 Tirosin-chinasi citoplasmatica

PDGFRBPDGFRAFGFR1C-KITMPLEGFRRecettori(Tirosin Chinasi)

TIROSIN-CHINASI

GENE CANDIDATO : JAK2

Mappa nella regione di LOH del 9p24Presente nel 30%-35% PV

Stimola: formazione di colonie in vitro differenziazione eritroide

risposta proliferativa all’EPO

VAL 617F in PVVAL 617F in PV

WT

PV

MUTAZIONI DI JAK2(Activating point V617F mutation)

Esone 12 JAK2:

G sostituita da una T (nucleotide 1849)

Valina sostituita da Fenilalanina (aminoacido 617)

Mutazione somatica: cellula staminale pluripotente eterozigote o omozigote attivazione costitutiva di JAK2

ST

AT

ST

AT

ST

AT

JAK

JAK-STAT PATHWAYJAK-STAT PATHWAY

NucleusNucleusCytosolCytosol STATSTAT

Plasma membranePlasma membrane

JAKJAK

ST

AT P P

P P

JAKJAKS

TA

T

ST

AT

ST

AT

ST

AT

ST

AT P

ST

AT

P

EPOEPO

EPOEPO

EPOEPO

EPOEPO

P

P

P

P

Ep

o-R

Ep

o-R

Ep

o-R

Ep

o-R

Ep

o-R

Ep

o-R

JAK

JAK-STAT PATHWAYJAK-STAT PATHWAY

NucleusNucleusCytosolCytosol STATSTAT

Plasma membranePlasma membrane

JAK-Val617FJAK-Val617F

ST

AT

ST

AT

ST

AT

ST

AT

ST

AT P

ST

AT

P

P

P

P

Ep

o-R

Ep

o-R

ST

AT

ST

AT

ST

AT

ST

AT P P

P P

Ep

o-R

Ep

o-R

JAK-Val617FJAK-Val617F

JAK-Val617FJAK-Val617F JAK-Val617FJAK-Val617F

P

CMLBCR/ABL1

Others

PV

MFET

PDGFRBPDGFRAKITFGFR1

Myeloproliferative Disorders

JAK2 MPL

CITOPLASMA

NUCLEO

ABL1

JAK2 Tirosin-chinasi citoplasmatica

PDGFRBPDGFRAFGFR1C-KITMPLEGFRRecettori(Tirosin Chinasi)

TIROSIN-CHINASI E NEOPLASIE MIELOIDI CRONICHE

Proliferation

RAS

GRB-2

SOS

PDGFRLigand PDGFR HLH

PDGFRAPDGFRB

Tyrosin kinase domain

PDGFRB

•Codificato dal gene PDGFRB localizzato sul cromosoma 5q33

•mRNA di 5.5 Kb

•Proteina costituita da 1067 amminoacidi

t(5;12)(q33;p13)-ETV6/PDGFR

Diagnosi: atypical CML CMML MDS/MPD

Eosinofilia BM/PB

Monocitosi PB>8%

Risposta al trattamento con Imatinib Mesilato

7q11/HIP1

14q22/NIN

14q32/KIAA1509

1p36?

17p11/HCMOGT-1

12p13/ETV6

17p13/RABEP1

14q32/CEV14

10q22/H4

16p13/NDE115q22/TP53BP1(RABPT5)

12q13/?

2q37/?

3p21/?

1q23/PDE4DIP

1q21/TPM3

PDGFRB

Geni partners di PDGFRB nei disordini Mieloproliferativi cronici Ph-

NH2 COOH

PDGFRBGene Partner

DUAL COLOUR FISH t(1;5)(q21;q33)/TPM3-PDGFRB

5

5’

3’

PDGFRBCTD-2601I11

der(1)

der(5)

CTD-2601I11 (PDGFRB) + RP11-205M9 (TPM3)

15

1

RP11-205M9

5’

3’

TPM3

Proliferation

RAS

GRB-2

SOS

PDGFRLigand PDGFR HLH

PDGFRAPDGFRB

Tyrosin kinase domain

RP11-3H20

FIP1L1 CHIC2 PDGFRA

telcen

telcenFIP1L1/PDGFRA

Hypereosinophilic Syndrome, Myeloproliferative Variant

4q12

I-FISH 34 pts with HES

FIP1L1/PDGFR+ FIP1L1/PDGFR-

PTS 8 26

M/F 7/1 12/14

Median age 42 51

Hepatomegaly 6 5

Splenomagaly 6 6

Hearth 2 3

CNS 2 3

Lung 1 3

Skin 4 10

Response toSTI571

7/7 0/4

1. conta degli eosinofili nel sangue persistentemente

superiore a 1,5 x109/l per un periodo superiore a sei

mesi

2. segni e/o sintomi di coinvolgimento di organi

3. assenza di una causa nota o di un’anomalia clonale

HES

I 3 criteri di definizione della HES in base alla WHO sono:

The 8p11 myeloproliferative syndrome8p11 MPS

Myeloid hyperplasia

Eosinophilia

Associated T-cell lymphoma

Progression toward AML

der(13)

der(8)813

t(8;13)(p11;q12)-ZNF198/FGFR1

Ig-like domainstransmembrane

tyrosin kinase

fusion partners

FGFR113q12/ZNF198

9q33/CEP110

6q27/FOP

22q11/BCR

N CIg1 Ig2 Ig3 TM TK1 TK2

19q13/HERV-K

11p15/?

17q25/? 12q15/?

12p11/FOP2

WHO Classification of Systemic Mastocytosis

Mast cell leukemia

Aggressive systemic mastocytosis

SM with an associated Haematopoietic clonal non-mast cell lineage disease

Indolent Systemic Mastocytosis ( Isolated bone marrow mastocytosis;

Smouldering systemic mastocytosis )

Variants and subvariants

FIP1L1 PDGFRACHIC2(Deletion)

KIT(Mutations)

5’ 3’ 3’5’centromero telomero

C-Kit activation loop: D816V (aspartic acid to valine at aa 816)

D816Y (aspartic acid to tyrosine at aa 816)

Resistence: imatinib mesilate (in vitro and in vivo)

Sensitivity: PK412 (in vitro)

Systemic mast cell disease / acute myeloid leukemia

C-KIT mutations

Mutation of kinase domain

Gastrointestinal stromal tumors(GIST)

C-KIT mutations

Mutation of extracellular domain

Mutation of juxtamembrane domain

PDGFRA mutations Mutation of kinase domain

Sensitivity: imatinib mesilate in vivo

somatiche germlineGIST sporadico GIST familiare

PDGFRA C-KIT

Seminoma

Sarcomi

Carcinoma polmonare a piccole cellule

Glioblastoma

Osteosarcoma

Sarcoma polmonare

Dermatofibrosarcoma

Tumori cerebrali

Kobayashi et al. NEJM 24/2/05

• A non-small-cell lung cancer that was highly responsive to gefitinib contained a mutation in the epidermal growth factor receptor (EGFR) gene that increases susceptibility of the tumor to gefitinib

• After two years of remission, the disease relapsed

• A second biopsy of the tumor revealed a new mutation in the gene that negated the effects of gefitinib

Summary of EGFR mutations (PNAS 2004 101 13306)

Mutation of HER2 in the Kinase domain is Activating

Wang et al. 2006 Cancer Cell 10 25

EGFR INHIBITORSEGFR INHIBITORS

Gefitinib

Erlotinib

Lapatinib

* Also inhibits VEGFR2

CMLBCR/ABL1

Others

PV

MFET

PDGFRBPDGFRAKITFGFR1

Myeloproliferative Disorders

JAK2 MPL

NDE1/PDGFRB FUSION NDE1/PDGFRB FUSION t(5;16)(q33;p13)t(5;16)(q33;p13)

PHILADELPHIA-NEGATIVEMYELOPROLIFERATIVE DISORDERS

• Polycytemia Vera• Essential Thrombocytemia• Chronic Idiopathic Myelofibrosis

• Chronic Eosinophilic Leukemia• Chronic Neutrophilic Leukemia• Chronic Myeloproliferative disease unclassifiable• Myelodysplastic / Myeloproliferative diseases• Systemic Mastocytosis, Mast Cell Leukemia

WHO HISTOLOGICAL CLASSIFICATION OFMYELODYSPLASTIC / MYELOPROLIFERATIVE

DISEASES

• Chronic Myelomonocytic Leukemia • Atypical Chronic Myeloid Leukemia • Juvenile Myelomonocytic Leukemia • Myelodysplastic / Myeloproliferative disease, unclassifiable

Ruolo delle proteine tirosin-chinasichenei disordini mieloproliferativi cronici: dalla diagnosi molecolare alla terapia mirata

2. Myelodysplastic/myeloproliferative diseases

-Chronic myelomonocytic leukemia

-Atypical chronic myeloid leukemia

-Juvenile myelomonocytic leukemia

- Myelodysplastc/myeloproliferative diseases, unclassifiable

1. Chronic myeloproliferative diseases

- Chronic myelogenous leukemia

- Chronic neutrophilic leukemia

- Chronic eosinophilic leukemia/ hypereosinophilic syndrome

- Polycythemia vera

- Chronic idiopathic myelofibrosis

- Essential thrombocythemia

- Chronic myeloproliferative disease, unclassifiable

1. Chronic myeloproliferative diseases

- Chronic myelogenous leukemia

- Chronic neutrophilic leukemia

- Chronic eosinophilic leukemia/ hypereosinophilic syndrome

- Polycythemia vera

- Chronic idiopathic myelofibrosis

- Essential thrombocythemia

- Chronic myeloproliferative disease, unclassifiable

Glivec

Glivec (imatinib mesilato) Meccanismo d’azione

Traslocazione 9;22

Ematopoiesi normale

Proteina di fusione Bcr-Abl

LMC

Proteina di fusione Bcr-Abl

MDS / MPD

t(5;12)(q33;p13)

MDS / MPD

t(5;12)(q33;p13)

PDGFRB gene

t(5;12)(q33;p13)

PDGFR gene

MDS / MPDCMML with eosinophilia

MPD Ph negative (eosinophilia; monocytosis)

t(5;12)(q33;p13)

PDGFR gene

HLH DNA-bindingN C

ETV6 gene

TYROSINE KINASE RECEPTOR FAMILY

Tyrosine Kinase domains

Cell Membrane

Extra Cellular

Intra Cellular

Dimerisation - Phosphorilation - Signal Transduction

Ligand binding