5a. tyrosine kinases
TRANSCRIPT
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