Protein Kinase + ADP O O. Protein OH + ATP Protein O P H 2 O. P i. Protein Phosphatase

Transcript

1 Protein Kinase O Protein OH + ATP Protein O P P i Protein Phosphatase H 2 O O O + ADP Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine, or tyrosine). A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein. A protein phosphatase catalyzes removal of the P i by hydrolysis.

2 Circa 500 serin- o treonin-kinasi, solo 96 Tirosin-kinasi in tutto il genoma umano. (funzioni importanti!)

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6 Meccanismi di attivazione delle TK Amplificazione iperespressione (ERB2, MET..) Geni di fusione (BCR-ABL, Tel-PDGFRb, FIP1L-PDGFRa ) Alterazioni strutturali interne (FLT3 ITD..) Mutazioni puntiformi (FLT3, JAK2, KIT..)

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10 Iperespressione Geni di fusione Attivazione costitutiva TK, guadagno di funzione e attività oncogenica ITD Mutazioni

11 Myeloid Leukemias are caused by a limited numbers of recurrent molecular defects: Stem Cell Myeloid Progenitor Proliferation Defects Differentiation defects CMPDs TK activation TK activation Acute Leukemias TFs involvement

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14 FLT3 alterations in AML TM JM TK1 K1 TK ITD D835 ITD and POIT MUTATIO ARE FREQUETLY FOUD I AML (36% of adult AML)

15 Relapse-free survival according to FLT3 (GIMEMA LAM-99P) ITD negative P= ITD positive Years from CR

16 KIT activation in AML Mutazioni presenti in circa il 5% delle LMA Associate a CBF leukemias Mutazioni in esoni diversi: D419X V560X D816X Valore prognostico negativo nelle t(8;21), ancora incerto nelle inv16 Iperespressione CD117 in circa il 20% delle AML

17 Iperespressione di CD117 Coexpressione di CD117 e di CD34

18 Prognosis poor poor??? Refs: Cairoli R e t al. Blood Schnittger S et al. Blood Boissel et al. Leukemia 2006 Paschka P et al. J Clin Oncol 2006

19 Mechanism of action ATP Bcr-Abl STI571 Bcr-Abl Substrate Substrate Y Y Imatinib = rivoluzione P P P Substrate Substrate P Y Effector Y Effector Y = Tyrosine P = Phosphate Druker B et al 1996

20 Per la prima volta si dimostrava che piccole molecole competitrici del sito di legame dell ATP delle TK: potevano essere abbastanza selettive e bloccare solo l attività di alcune TK; il blocco dell attività TK era efficace come attività antitumorale senza produrre effetti tossici gravi.

21 Risultati terapia con inibitori TK nelle neoplasie ematologiche Buoni, talora ottimi, nei disordini mieloproliferativi semplici (LMC, HES, CMML etc ) Molto inferiori e transitori nelle forme avanzate di trasformazione e nelle LA

22 Event-free Survival and Survival Without AP/BC on First-line Imatinib Progression events: 6.5% AP/BC 4.7% loss of MCyR 2.5% loss of CHR 1.4% CML-unrelated deaths Estimated rate at 54 months (with 95%CI) 84% (80-87) 93% (90-96)

23 Overall Survival on First-line Imatinib (ITT principle) 95% 89% Estimated rate at 60 months (with 95%CI) CML-related deaths All deaths 4.6% (2-7) 10.6% (8-14) Survival without CML-related deaths

24 Survival Without AP/BC by Molecular Response at 12 months on First-line Imatinib Estimated rate at 54 months n= % n= 94 95% n= % } } p=0.007 p<0.001

25 Survival Without AP/BC at 60 months by Level of CyR at 12, 18 and 24 months % without AP/BC at 60 months CCyR PCyR o MCyR % with CCyR after landmark months 18 months 24 months

26 Kantarjian H. et al. Clin Cancer Res 2004;10:68 75.

27 Gimema CML WP Study 002 Late CML unresponsive or intollerant to IF Progression-free survival The 80 TKI Imatinib per se seems Months to decrease the progression rate of the CML clone, probably decreasing its genomic instability

28 Imatinib DA damage ROS Radical Oxigen Species

29 Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph1 Ph+ STUDIO 106 OVERALL SURVIVAL Ph+ Studio 102 OVERALL SURVIVAL All deaths Ph+ Ph1 Ph1 Ph+ Ph+ Ph Median survival 90 Ph1 Ph1 Untreated Ph1patients 7.5 Ph+ months 90 Pretreated patients 5.6 months Ph+ Ph1 Ph1 Ph+ Ph+ Ph+ Ph+ 80 Since diagnosis of blast crisis 9.9 months p= Imatinib IF + Ara-C CML related deaths Ph+ Ph1 Ph1 50 Deaths after BMT 3 5 Ph Other deaths 8 7 Ph+ Ph+ Estimated survival Ph at 18 months (p=0.16) 97.2% 95.1% Ph+ Ph1 Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph1 Ph+ Ph1Ph+ Ph1 Ph1 Imatinib IF + Ara-C Imatinib IF + Ara-C Ph Months since start of treatment Months since randomization Ph1 % alive % of patients alive

30 CLIICAL TRIALS WITH FLT3 IHIBITORS CEP-701 (Cephalon) PKC412 (ovartis) SU5416 (Pharmacia) (ML518) (Millenium) SU11248 (Pfizer) L (Merck)

31 CEP701

32 ASH % remissioni complete % nei FLT3 mutati

33 Frequency of imatinib resistance within 3 years Percent Primary resistance Relapse/ progression Early chronic phase 4 Late chronic phase Accelerated phase (600 mg/d) Blastic phase (600 mg/d) Hochhaus and La Rosée Leukemia. 2004

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35 Difetto genetico semplice fenotipo leucemico Efficacia TKIs (..e non è solo questione di potenza!) Più difetti -> fenotipo leucemico complesso

36 16/199 8%

37 1.0 Dasatinib 70 mg BID in CP-CML Progression-free survival Proportion progression-free Intolerant Resistant Total o. progressed Months Progression was defined as confirmed AP / BP, loss of CHR / MCyR, or increasing WBC count

38 Proportion progression-free Dasatinib in blast-phase CML Progression-free survival CML-MB CML-LB o. of deaths Median (mo) Updated ASH Months Patients who underwent SCT were censored at that time

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40 Resistenza = fenomeno plastico Clone without mutations Imatinib Clone with Mutation LBH or O Clone with T315I Dasatinib or nilotinib

41 at Rev Cancer, Dec 2006 Genetic and epigenetic alterations that are beneficial to a neoplastic clone are facilitated by natural selection and genetic drift The fitness of a neoplastic cell is shaped by its interactions with cells and other factors in its microenvironment (its ecology), including interventions to prevent or cure cancer.

42 Genetic Instability may increase the evolution speed Acquired From the beginning

43 Blood 2007

44 I TKIs non sembrano in grado da soli di eliminare le cellule staminali leucemiche Molecular response in IRIS trial Log reduction of BCR-ABL Base line ot detected CCR Pre Months 92% BCR- ABL positive

45 Dynamics of CML response to imatinib Michor et al., ature 2005 Differentiated Progenitors

46 Primitive quiescent BCR-ABL+ leukemic stem cells are less sensitive to imatinib Holyoake TL, Blood 2002

47 Holyoake TL, Blood 2004 Punish the Parent not the Progeny In CML progenitor cells (lin-, CD34+, CD38- cells): A) Decreased Imatinib concentration: - decreased influx (OCT-1) (White DL et al., Blood 2006) - increased efflux (ABCB1/MDR1, ABCG2) Bcr-Abl off? (Zhou et al, 2001) Bcr-Abl on? (Burger et al, 2004) (Jordanides et al., 2006) B) Increased expression of BCR-ABL

48 Blood 2006

49 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 Imatinib Ph+ cells BCR-ABL inhibition Ph+ cells that survive and return to normality

50 Significant Improvement in Rate of CMR with Continuous Imatinib Therapy Months of Imatinib Therapy 24 months 36 months 48 months 69 months Branford et al. Blood, 108 (11): Abstract 430

51 Rousselot et al., Blood 2007

52 01/11/00 01/03/01 01/07/01 01/11/01 01/03/02 01/07/02 01/11/02 01/03/03 01/07/03 01/11/03 01/03/04 01/07/04 01/11/04 01/03/05 Can cure be achieved with imatinib alone? ,1 occr CCR SMR Pazient CS: RQ PCR analysis on PB and Bone Marrow samples Imatinib 400 mg Imatinib stop (30 months) GMR 0,01 0,001 0,0001 ested PCR-negative

53 Why? Stem cell senescence? Reactivation of an autologous immune control on the CML stem cells?

54 The strategy to eradicate the persistence of this tricky Ph+ population must be appropriate Immunotherapy? Specific targeted therapies for stem cells? Vaccines?

55 Conclusioni I TKIs sono entrati nell armamentario terapeutico delle neoplasie ematologiche e lo hanno arricchito Sono molto efficaci nei CMPDs, meno nelle forme avanzate di malattia e nelle forme acute I meccanismi di resistenza appaiono molto più complessi di quanto originariamente immaginato. Per le forme avanzate ed acute appare

56 San Luigi Hospital-University of Turin 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

57 ROS01001.PPT v CML GIMEMA WP