Solo 96 TKs in tutto il genoma umano!

GRB2 GAB2 SOS RAS GDP SHC GTP ERK JUN Nucleus

SHC GRB2 GAB2 PI3K mTOR Apoptosis block

Perché esiste una specificità di risposta se tutti i segnali attivano le stesse vie? Perché gli stessi segnali possono attivare risposte diverse?

Radiatons->genetic damage ABL is a “shuttle” TK Radiatons->genetic damage ABL NUCLEUS Cell Cycle Rb, p53,p73 ABL Block in G1

Scaffold protein

Intensità del segnale Durata del segnale Possibilità di spegnerlo più o meno rapidamente

Meccanismi di attivazione Riarrangiamenti Amplificazioni Mutazioni puntiformi ……….

Riarrangiamenti Spesso conseguenza di traslocazioni cromosomiche (BCR-ABL, NPM-ALK) o di delezioni intracromosomiche (FIP1-PDGFRa) Meccanismo più frequente nelle neoplasie ematologiche che nei tumori solidi

Autophosphorylation by dimerization Phosphorylation of substrates ABL ABL Tyr Tyr BCR BCR Phosphorylation of substrates P

Amplificazioni Più frequenti nei tumori solidi che nei tumori ematologici (ERB2 nel Ca mammario etc…) Più frequente per recettori di membrana Meccanismo di attivazione: l’aumento delle molecole in superficie ne favorisce il contatto anche in assenza di ligando

Mutazioni puntiformi Numerosi esempi sia nei tumori ematologici (JAK2 in DMPC-Ph-negativi) che nei tumori solidi (KIT nei GIST) Meccanismo di attivazione: meccanismo di attivazione diretto del dominio TK P * amino acid substitutions

FLT3 activation in AML (30% of cases) ITD P P P D835Y STAT JAK RAS

Radiatons->genetic damage ABL is a “shuttle” TK Radiatons->genetic damage BCR/ABL ABL NUCLEUS Cell Cycle Rb, p53,p73 ABL Block in G1 Deacreased DNA repair= genomic instability Jane Wang, Paolo Vigneri et al.

Imatinib Mechanism of action Bcr-Abl Proteina Tir ATP binding pocket STI P ATP

Imatinib Response and Disease Phase Chronic phase IFN failure (n=532) Accelerated phase (n=235) Myeloid Blast crisis (n=229) Haematological response 95% 83% 31% Complete 54% 8% No evidence of leukaemia - 12% 4% Return to CP 17% 18% Major cytogenetic response 60% 26% 15% 41% 7% Partial 19%

N N N Ph+ N N N N N N N N N N N Ph+ Ph+ N N N N N N Ph+ N N N N N N Ph+ Ph+ Ph+ N N N N N N N N N Ph+ Ph+ N N N N Ph+ N N N N Ph+ Ph+ Ph+ N N N N N Ph+ N N Ph+ Ph+ N N Ph+ N N Ph+ N N Ph+ Ph+ N N Ph+ N Ph1 Ph+ N Ph+ Ph+ N N N N Ph+ Ph+ Ph1 Ph1 N Ph+ N N Ph+ Ph1 Ph+ N Ph1 Ph1 N Ph+ Ph1 Ph1 Ph+ Ph+ N Ph+ Ph1 Ph+ Ph1 Ph1 Ph+ Ph+ Ph+ Ph+ Ph1 Ph+ Ph1 Ph+ Ph+ Ph+ Ph1 Ph1 Ph1 Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph1

Cumulative Best Response at 12 and 60 months on First-line Imatinib in IRIS study 96% 96% 98% 98% 92% 92% 85% 85% 87% 69% 69% Complete hematologic response (CHR) = white cell count of <10 x 109, platelet count of <450 x 109, <5% myelocytes plus metamyelocytes, absence of blasts and promyelocytes from peripheral blood, absence of extramedullary involvement, and no signs of accelerated-phase (AP) or blast-crisis (BC) phase disease Major cytogenetic response (MCyR) = 1%-35% Philadelphia-chromosome–positive (Ph+) metaphases Complete cytogenetic response (CCyR) = absence of Ph+ metaphases Cumulative rates of CHR and CCyR were estimated according to the Kaplan-Meier method, in which patients who crossed over to the other treatment arm or discontinued treatment for reasons other than progression were censored at the last follow-up visit of the initial treatment period Cumulative estimated response rates increased over time for CHR, MCyR, and CCyR. Late responses are seen An estimated 69% patients achieved CCyR at 12 months Of those patients who did not achieve a CCyR at 12 months (31%), 60% went on to achieve a response Time (months) CHR (%) MCyR (%) CCyR (%) 12 96 85 69 18 97 88 76 24 90 80 60 98 92 87

Event-free Survival and Survival Without AP/BC on First-line Imatinib 98% 84% Estimated rate at 60 months (with 95%CI) 93% (90-96) 83% (80-87) Actual Events 6.3% AP/BC (n=35) 5.1% loss of MCyR (n=28) 2.5% loss of CHR (n=14) 1.6% CML-unrelated deaths (n=9) Events were defined by the first occurrence of any of the following: death from any cause during treatment, progression to AP or BC phase of CML, loss of CHR, loss of MCyR, or increasing white blood cells (WBC) 9 CML-unrelated deaths were recorded for patients on imatinib treatment (with no signs of clinical progression). The recorded events included: cardiac arrest (2), cardio-respiratory arrest, metastases to liver, pulmonary edema & acute renal failure, road traffic accident, metastatic rectal cancer, sepsis, and myocardial infarction. 86 (15.5%) events were observed during treatment with imatinib. This resulted in an estimated event-free survival of 83% at 60 months. Estimated rate of survival without progression to AP/BC at 60 months is 93%

Annual Event Rates on First-line Imatinib Year All events* AP/BC 1st 3.3% 1.5% 2nd 7.5% 2.8% 3rd 4.8% 1.6% 4th 1.5% 0.9% 5th 0.9% 0.6% Annual rates of all events as well as progression to AP/BC decline over time with imatinib therapy * All deaths or loss of response including progression to AP/BC

Survival Without AP/BC by Molecular Response at 12 months on First-line Imatinib Estimated rate at 60 months  n=136 100% n= 94 95% n=138 88%  CCyR with >=3 log red. p=0.007 Molecular response was evaluated every 3 months after a CCyR was obtained using real-time quantitative polymerase chain reaction (RQ-PCR) The ratio of BCR-ABL:BCR transcripts was measured Results were expressed as “log reductions” below a standardized baseline derived from a median BCR-ABL:BCR value obtained from 30 untreated patients with chronic-phase CML Major molecular response (MMR) was defined as ≥3 log reduction of BCR-ABL:BCR transcripts No patient with CCyR and MMR by 12 months progressed to AP/BC CCyR with <3 log red. p<0.001 No CCyR Druker et al., ASCO 2006

The BCR/ABL amount measured by RQ PCR mirrors the number of cells less sensitive to Imatinib Imatinib Imatinib Ph+ BCR/ABL reduction PhR PhR The persistent Ph-positive cells less sensitive to imatinib may potentially become prone to progression Higher the number, higher the risk!

Two major problems with imatinib therapy Resistance/Loss of Response and Progression Persistence of small amount of leukemic cells

to suppress the BCR-ABL TK Pathophysiology of imatinib resistance Pathophysiology of imatinib resistance BCR-ABL proliferation Other defects Imatinib BCR-ABL proliferation Imatinib not able to suppress the BCR-ABL TK Clonal evolution Point mutations BCR-ABL amplification Insufficient IMA in cells

a.a. that control the kinase activation step The map of mutations 4 Critical regions a.a. that control the kinase activation step ATP binding loop P-loop Gate keepers Catalytic domain Activation loop M244V D276G V289A M343T E355G/D H396R/P S417Y L248V T277A M351T/V L387M/F G250E E255K/V F311L/I F317L F359V F382L E459K Q252R/H F486S V379I Y253F/H T315I A380T

Most frequent mutations P-loop 22 21 20 19 18 17 16 15 14 13 12 no. of mutations 11 10 9 8 7 6 5 4 3 2 1 M244V L248V G250E Q252R/H Y253F/H E255K/V D276G T277A P296H F311L/I T315I F317L M343T M351T/V E355G/D F359V/I V379I A380T F382L L387F/M H396R/P S417Y E459K/Q F486S *Soverini et al GIMEMA-CML WP data on 297 matinib resistant patients (CML, Ph+ ALL)

Soverini et al., JCO 2005

Dual Inhibitors Imatinib derivative

Wild-type Wild-type T3151 Mutant T3151 Mutant Gorre et al., Science. 2001

Clone without mutations Clone with Mutation Clone with T315I Imatinib Clone without mutations Clone with Mutation LBH or ON012380 Clone with T315I Dasatinib or nilotinib

Imatinib Imatinib Ph+ Ph+ Ph+ PhU Ph+ PhU Ph+ Ph+ Ph+ Ph+ PhU PhU PhU PhR PhU Ph+ Ph+ PhR Ph+ Ph+ Ph+

Blood 2006

Molecular response in IRIS trial Base line 1.0 CCR Log reduction of BCR-ABL 2.0 3.0 92% BCR-ABL positive The vast majority of pts rapidly had levels of residual leukaemia that were below the level of detection by cytogenetic analysis 92% of pts have detectable levels of BCR-ABL up to 30 months 4.0 Not detected Pre 3 6 9 12 18 24 27 30 Months

CML cells Imatinib Less sensitive progenitor Ph+ progenitors Normal SC Ph+SC Ph+SC Ph+SC Normal SC Normal SC Normal SC Normal SC Normal SC CML cells Imatinib Ph+ mature cells

Most patients who stop imatinib therapy, even when PCR neg, relapse!

Michor et al., Nature 2005 Differentiated Progenitors

Punish the Parent not the Progeny The Imatinib concentration in the progenitor cells, due to the action of influx (OCT-1) and efflux (ABCB1) proteins seems to represent a major determinant for the persistence-resistance of Ph-positive progenitors Tessa Holyoake and Deb White Bcr-Abl off? Bcr-Abl on? Holyoake TL, Blood 2004

Ph+ cells that survive and return The persistence of Ph-positive cells may be due to: - cells more resistant to imatinib - cells in which the BCR-ABL TK activity may be suppressed without a great damage The persistence of Ph-positive cells may be due to: - cells more resistant to imatinib - cells in which the BCR-ABL TK activity may be suppressed without a great damage Imatinib BCR-ABL inhibition Apoptosis Ph+ cells Imatinib BCR-ABL inhibition Ph+ cells that survive and return to “normality”

The strategy to eradicate the persistence of this tricky Ph+ population must be appropriate Combination therapy? Immunotherapy?

San Luigi Hospital-University of Turin Daniela Cilloni Giovanna Rege Cambrin Francesca Messa Carmen Fava Francesca Arruga Ilaria Defilippi Emanuela Messa Alessandro Morotti Enrico Gottardi Emilia Giugliano Anna Serra Milena Fava San Luigi Hospital-University of Turin

ICSG on CML