TIROSIN-CHINASI

Recettori transmembrana per fattori di crescita

Enzimi citoplasmatici
 TIROSIN-CHINASI

CITOPLASMA

PDGFRB
PDGFRA
ABL1 FGFR1
C-KIT
JAK2 MPL
Tirosin-chinasi citoplasmatica
EGFR
Recettori
NUCLEO (Tirosin Chinasi)
 JAK 2
MPL
RECEPTOR FAMILY
ARG (ABL2)
FLT3
Extra Cellular
KIT
PDGFRα
PDGFRβ
FGFR1

Cell Membrane

Tyr Kinase Intra Cellular

domains

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

• LEUCEMIA MIELOIDE CRONICA:

Ph-positiva
Ph-negativa, BCR-ABL 1
positiva
•
• Ph-negativa
 Myeloproliferative Disorders

Trombocitemia essenziale

Mielofibrosi idiopatica ET
MF

Policitemia vera PV

Others
CML
BCR/ABL1
 TIROSIN-CHINASI

CITOPLASMA

PDGFRB
PDGFRA
ABL1 FGFR1
C-KIT
JAK2 MPL
Tirosin-chinasi citoplasmatica
EGFR
Recettori
NUCLEO (Tirosin Chinasi)
 BCR/22q11 ABL1/9q34

Ph
BCR/ABL1

t(9;22)(q34;q11)
 CITOPLASMA

Apoptosi

BCR ABL1 Adesione Cellulare

Tirosin Chinasi

Proliferazione
NUCLEO
 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

GLIVEC
ATP Proteina
Proteina Bcr-Abl
Bcr-Abl
Substrato
Substrato
P
P
P Y
Y

Substrato
Substrato
P Y
Y
Effettore Y = Tirosina
Effettore
P = Fosfato
Goldman JM, Milo JV, NEJM 2001, 344:1084-
1086
Inibitori Tirosin chinasi

• Imatinib
• Nilotinib
• Dasatinib
• Sumitinib
• …………
• …………
 Studio IRIS Studio IRIS
Probabilità di risposta ematologica Probabilità di risposta citogenetica
completa maggiore
(stima a 12 mesi) (stima a 12 mesi)
% risposta % risposta
100 96 100
90 GLIVEC 90
80 %* 80 83
70 67 70 GLIVEC%
60 IFN- + 60
50
Ara-C
% 50 *
40 40
30 30 20
20 20 IFN- + %
10 10
0 0 Ara-C
0 3 6 9 12 15 18 21 0 3 6 9 12
Mesi dall’inizio del
* p<0,001 Mesi dall’inizio
trattamento
del trattamento

Risposta ematologica completa Risposte citogenetiche

Leucociti < 10 x 109/L Risposta citogenetica completa

Piastrine < 450 x 109/L 0% Ph+
Assenza di blasti, basofili < 20% Risposta citogenetica parziale
Assenza di malattia extramidollare 1-35% Ph+
 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
 Myeloproliferative Disorders

MPL
JAK2
ET
MF

PV

Others
CML
BCR/ABL1
 TIROSIN-CHINASI

CITOPLASMA

PDGFRB
PDGFRA
ABL1 FGFR1
C-KIT
JAK2 MPL
Tirosin-chinasi citoplasmatica
EGFR
Recettori
NUCLEO (Tirosin Chinasi)
 GENE CANDIDATO : JAK2

Mappa nella regione di LOH del 9p24 Stimola: formazione di colonie in vitro
Presente nel 30%-35% PV differenziazione eritroide
risposta proliferativa all’EPO
VAL 617F in PV

PV

WT
 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
 JAK-STAT PATHWAY
EPO EPO

EPO EPO

Epo-R
Epo-R
Epo-R
Epo-R
Epo-R

Epo-R

Plasma membrane

STAT

STAT
P P

P P JAK JAK

STAT
STAT
P P
JAK JAK STAT

P P
JA
K
JA

STAT

STAT
P
STAT

STAT

STAT
P
STAT
Cytosol Nucleus
 JAK-STAT PATHWAY

Epo-R
Epo-R
Epo-R
Epo-R Plasma membrane

STAT

STAT
P P

P P JAK-Val617F JAK-Val617F

STAT

STAT
JAK-Val617F JAK-Val617F P P

P P
STAT
STAT

STAT
P
STAT

STAT

STAT
P
STAT
Cytosol Nucleus
 Myeloproliferative Disorders
MPL
JAK2
ET
MF

PV

Others
PDGFRB CML
PDGFRA BCR/ABL1
KIT
FGFR1
TIROSIN-CHINASI E NEOPLASIE MIELOIDI CRONICHE

CITOPLASMA

PDGFRB
PDGFRA
ABL1 FGFR1
C-KIT
JAK2 MPL
Tirosin-chinasi citoplasmatica
EGFR
Recettori
NUCLEO (Tirosin Chinasi)
 Ligand PDGFR HLH
PDGFR

Tyrosin kinase domain

GRB-2

SOS

RAS
PDGFRA
PDGFRB Proliferation
 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

Geni partners di PDGFRB nei disordini Mieloproliferativi
cronici Ph-

7q11/HIP1
3p21/? 10q22/H4
2q37/? 12p13/ETV6

1p36? 12q13/?
PDGFRB 14q22/NIN
1q23/PDE4DIP
14q32/CEV14
1q21/TPM3
14q32/KIAA1509
17p11/HCMOGT-1 15q22/TP53BP1(RABPT5)
17p13/RABEP1 16p13/NDE1

NH2 COOH

Gene Partner PDGFRB

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

3’
CTD-2601I11 (PDGFRB) + RP11-205M9 (TPM3)
TPM3 RP11-205M9

5’
der(1)

1 der(5)

1
5
3’

PDGFRB
CTD-2601I11
5’

5
 Ligand PDGFR HLH
PDGFR

Tyrosin kinase domain

GRB-2

SOS

RAS
PDGFRA
PDGFRB Proliferation
Hypereosinophilic Syndrome, Myeloproliferative Variant

FIP1L1 CHIC2 PDGFRA

4q12 cen tel
RP11-3H20

cen tel
FIP1L1/PDGFRA
 I-FISH 34 pts with HES

FIP1L1/PDGFR+ FIP1L1/PDGFR-

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 to 7/7 0/4
STI571
 HES

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

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

The 8p11 myeloproliferative syndrome
8p11 MPS

Myeloid hyperplasia
Eosinophilia
Associated T-cell lymphoma
Progression toward AML
t(8;13)(p11;q12)-ZNF198/FGFR1

der(13)
13
8 der(8)
 fusion partners

9q33/CEP110
6q27/FOP 11p15/?

22q11/BCR 13q12/ZNF198
FGFR1
19q13/HERV-K 12p11/FOP2
17q25/? 12q15/?

Ig-like domains tyrosin kinase

transmembrane

N Ig1 Ig2 Ig3 TM TK1 TK2 C

 WHO Classification of Systemic Mastocytosis

Variants and subvariants

Indolent Systemic Mastocytosis

( Isolated bone marrow mastocytosis;

Smouldering systemic mastocytosis )
SM with an associated Haematopoietic
clonal non-mast cell lineage disease

Aggressive systemic mastocytosis

Mast cell leukemia

5’ 3’ 5’ 3’
centromero telomero
FIP1L1 CHIC2 PDGFRA KIT
(Deletion (Mutations)
 C-KIT mutations

Systemic mast cell disease / acute myeloid leukemia

Mutation of kinase domain

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)

 Gastrointestinal stromal tumors
(GIST)

Mutation of extracellular domain

C-KIT mutations
Mutation of juxtamembrane domain
somatiche germline
GIST sporadico GIST familiare

PDGFRA mutations Mutation of kinase domain

Sensitivity: imatinib mesilate in vivo

 PDGFRA C-KIT

Glioblastoma Seminoma

Osteosarcoma Sarcomi

Sarcoma polmonare Carcinoma polmonare a piccole cellule

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 INHIBITORS

Gefitinib

Erlotinib

Lapatinib

* Also inhibits VEGFR2

 Myeloproliferative Disorders
MPL
JAK2
ET
MF

PV

PDGFRB
PDGFRA
Others
KIT
CML
FGFR1
BCR/ABL1
NDE1/PDGFRB FUSION t(5;16)(q33;p13)
 PHILADELPHIA-NEGATIVE
MYELOPROLIFERATIVE 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 OF

MYELODYSPLASTIC / MYELOPROLIFERATIVE
DISEASES
• Chronic Myelomonocytic Leukemia
• Atypical Chronic Myeloid Leukemia
• Juvenile Myelomonocytic Leukemia
• Myelodysplastic / Myeloproliferative disease, unclassifiable
Ruolo delle proteine tirosin-chinasiche
nei 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 (imatinib mesilato)
Meccanismo d’azione
Glivec
Traslocazione 9;22

Proteina di fusione Bcr-Abl Proteina di fusione Bcr-Abl

Ematopoiesi
LMC normale
 MDS / MPD

t(5;12)(q33;p13)
 MDS / MPD
t(5;12)(q33;p13)

PDGFRB gene
 MDS / MPD
CMML with eosinophilia

t(5;12)(q33;p13)

PDGFR gene
t(5;12)(q33;p13)
MPD Ph negative
(eosinophilia; monocytosis)

N HLH DNA-binding C

ETV6 gene

PDGFR gene
TYROSINE KINASE Ligand binding
RECEPTOR FAMILY

Extra Cellular

Cell Membrane

Tyrosine Kinase Intra Cellular

domains

Dimerisation - Phosphorilation - Signal Transduction