Molecular pathogenesis of CML: Recent insights

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1 Napoli 24 gennaio 2012 Molecular pathogenesis of CML: Recent insights Giuseppe Saglio University of Turin

2 CML Biology CML Clinical practice

3 MILESTONES IN MOLECULAR BIOLOGY OF CML Nowell P.C. & Hungerford D.A Konopka J.B. et al. Protein BCR-ABL Groffen J. et al Shtivelman E Consequence: new BCR-ABL fusion proteins with a constitutive TK activity

4 MILESTONES IN MOLECULAR BIOLOGY OF CML Daley G. Q. et al. Induction of CML in vivo Druker B. et al. Targeted therapy with TK inhibitors

5 Ph-chromosome, translocation t(9;22) Leukemia Phenotype Incidence CML >95% ALL»25-30% (children»5%) AML» 2% lymphomas sporadic

6 The different breakpoint positions within BCR lead to the formation of BCR-ABL transcripts and proteins with different BCR portions joined to the same ABL portion BCR ABL P190 BCR ABL P210 (e13a2) BCR ABL P210(e14a2) BCR ABL p230

7 BCR-ABL proteins and leukemia phenotype P190 P210 P230 Acute (Lymphoid) Leukemia. Chronic Myeloid Leukemia Chronic Neutrophilic Leukemia Why? Activation of specific pathways?

8 CML progression Chronic Phase Accelerated Blastic Phase»100% of the cases Could we define them BCR-ABL related diseases?»20-30% lymphoid

9 CML = in origin, a single basic defect Activation ofpathways substainingabnormal proliferation Activation of antiapoptotic pathways Always the truth? BCR-ABL Genomic instability Stem cell maintenance

10 The genomic instability of the Ph-positive clone is the key factor for progression Is the BCR-ABL rearrangement the cause or the consequence of this instability?

11 years N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 5 N N N N N N N Additional genetic defects N N N N - p53 inactivation N N N N N N N N N N N - p16 inactivation N N N N N -NRb Ninactivation N N N Ph1 N - NEVI Noverexpression N blast crisis N Ph1 Ph1 N Ph1 N -..many N others Ph1 Ph1 Ph1 N Ph1 Ph1 N Ph1 Ph1 Ph1 Ph1 Ph1 Ph1 Ph1 Ph1 Ph1 Ph1 Ph1 Additional defects include: Increasing Imatinib Resistance

12 BCR-ABL increases ROS production through the activation of PI3K/Akt pathway BCR-ABL Genomic instability Stem cell maintenance Oxidative stress

13 Bcr-Abl induces reactive oxygen species that cause self-mutagenesis Koptyra M et al. Blood. 2006;108: BC = blast crisis; Bcr-Abl = oncogenic tyrosine kinase; CP = chronic phase; N = healthy volunteers.

14 ROS toxicity on a cellular level ROS OH. OH radicals are highly reactive and can modify DNA, proteins and lipid components of cells H 2 O 2 O2 O 2 Lipid peroxidation Oxidations: CO, met, tyr Base oxidation Lysosomes Protein DNA ROI are normally converted to water by resident enzymes SOD and GPX Mitochondrion endoplasmic reticulum GPX, glutathione peroxidase; ROI, reactive oxygen intermediates

15 Fialkow s hypothesis Clonal Expansion Stem Cell Unknown genetic lesion causing genomic instability BCR/ABL rearrangement Ph-positive Stem Cell Other Cytogenetic abn. (Ph-negative cell) Myeloid Myeloid Lymphoid Lymphoid T-cell Ph-positive clonal B-cells Ph-negative clonal B-cells T-cell

16 Reports of clonal cytogenetic abnormalities in the Ph-negative cell population : 1) Meunier et al. Blood ) Bumm T et al., Blood ) Braziel et al., Blood ) O Dwyer et al, Leukemia 2003 et al - 5-7% of patients in CCyR after Imatinib Rx - in some cases, morphological or clinical features of MDS

17 The occurrence of a BCR-ABL rearrangement seems a likely and frequent event per se, maybe further facilitated by genomic instability Very low amounts of Bcr/Abl transcripts have been detected also in normal subjects. Biernaux et al., Blood 1995 Bose et al., Blood 1998 The formation of Bcr/Abl fusion genes can be easely induced in vitro in negative cell lines by exposing them to ionizing radiations Deininger MW et al., Cancer Research 1998

18 BCL2 BCl-xL Genetic predisposion HSC B Genetic lesions B Leukemic SC Increased survival Blood :

19 Helia Neves et al., Blood 1999

20 Saglio G. et al. PNAS 2002

21 Previous therapy of CML Conventional treatment hematological remission Allogeneic BMT IFN-a Ph-positive clone suppression or eradication The achievement of a good cytogenetic and molecular remission (PCR-negativity) is associated with an increased survival in CML

22 Conventional chemo-therapy à only sporadic cytogenetic responses no big survival advantages α-ifn therapy à consistent percentage of good cytogenetic responses (30%) àsurvival advantage % survival IFN p=0, CHT years ICSG on CML Prot. CML/86

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

24 CML Survival After Imatinib Introduction (MD Anderson Experience) Kantarjian H. et al Blood Mar 1;119(9):1981-7

25 Overall Survival of CML patients: still an issue? Overall Survival - Imatinib Arm Alive, % Survival: deaths associated with CML Overall survival Months since randomization Estimated overall survival at 8 years was 85% (93%, considering only CML-related deaths) Deininger M et al. Blood (ASH Annual Meeting Abstracts), Nov 2009; 114: 1126

26 IRIS 8-Year Update: Majority of Events Occur Early % with event Event Loss of CHR, Loss of MCyR, AP/BP, Death during treatment AP/BP Year Hughes et al; Blood : Deininger M, et al. ASH 2009; 114: Poster #

27 Data cut-off: 27Jul2011. Clark RE, et al. Haematologica. 2012;97(s1):237 [abstract 0583]. Survival After Progression to AP/BC (ENESTnd and IRIS) ENESTnd 3-Year Update ENESTnd IRIS % Alive Median survival ~10.5 months Months Since Progression

28 CLONAL EVOLUTION IN AML: DIAGNOSIS TO RELAPSE For CML: Diagnosis Response & Relapse Ding et al. Nature 401:506, 2012

29 Number of Leukaemic cells Propensity to progress

30 Simplified Vision of Resistance to Imatinib Imatinib is no more able to suppress the BCR-ABL TK Imatinib still works, but. Other defects Point mutations BCR-ABL amplification Insufficient IMA in cells Clonal evolution (with or without ACA)

31 Can a stronger inhibition of the BCR-ABL TK produce better results? Nilotinib BcrAbl > PDGFR > Kit > Src (Phos. IC 50 ) 19 nm 75 nm 209 nm >1000 nm Imatinib PDGFR > Kit > BcrAbl > Src (Phos. IC 50 ) 72 nm 99 nm 192 nm >1000 nm Dasatinib Src > BcrAbl > PDGFR > Kit (Phos. IC 50 ) 0.1 nm 1.8 nm 2.9 nm 18 nm Nilotinib has no significant effect on other kinases evaluated, including Src, FLT3, VEGFR, EGFR, InsR, RET, MET, IGFR at concentrations <3000 nm. Mestan. Blood a: Abs 1978, 2004 Weisberg. Cancer Cell 7:129, 2005

32 BCR-ABL1 transcript levels at 3 months are the only requirement for predicting outcome for CML patients treated with TKIs Marin et al. J Clin Oncol Jan 20;30(3):232-8.

33 Why kinetics of response is so important? Months % BCR-ABL% 10% 1% 0,1%

34 3 mo <10% BCR-ABL 3 mo >10% BCR-ABL Diagnosis TKIs How can we reduce this component? These are the patients in which an intervention is justified Longer persistance, higher risk of progression

35 ELN, OPTIMAL RESPONSE 2009* months - CHR, and - At least minor CgR (Ph + 65%) 6 months - At least PCgR ( < 35%) - CHR, and - At least PCgR (Ph + < 35%) and / or - BCR-ABL < 10% - CCgR (Ph + 0), and / or - BCR-ABL < 1% 12 months - CCgR (Ph + 0) - MMR (BCR-ABL 0.1%) THEN - MMR (MR 3.0) or better - MMR (MR 3.0) or better Baccarani M et al., J Clin Oncol., 2009; 27: Baccarani em et al, submitted

36 ENESTnd 5-Year Update Cumulative Incidence of MMR Nilotinib 300 mg BID (n = 282) Nilotinib 400 mg BID (n = 281) Imatinib 400 mg QD (n = 283) By 1 Year a By 4 Years a By 5 Years a Patients With MMR, % %, P < %, P <.0001 Δ 24% to 28% 27% 76%, P < %, P <.0001 Δ 17% to 20% 56% 77%, P < %, P <.0001 Δ 17% 60% MMR, major molecular response (BCR-ABL IS 0.1%). a Cumulative response rates reported consider each year to consist of twelve 28-day cycles. Time Since Randomization, Calendar Years 36 Data cutoff: May 22, 2013

37 OS by BCR-ABL Levels at 3 Months ENESTnd 5-Year Update Nilotinib 300 mg BID Imatinib 400 mg QD EMR Failure: 9% of pts EMR Failure: 33% of pts Patients Alive, % BCR-ABL Level 1% > 1% to 10% > 10% Censored Observations Pts Evt Cen OS by 5 Years a 97.6% 95.7% Time Since Randomization, Calendar Years 81.9% P =.4871 P =.0007 Patients Alive, % BCR-ABL Level 1% > 1% to 10% > 10% Censored Observations Pts Evt Cen Time Since Randomization, Calendar Years OS by 5 Years a 99.2% 95.3% 79.5% P =.0873 P <.0001 Patients with EMR failure (BCR-ABL > 10% at 3 months) have significantly worse 5-year OS Rates of EMR failure are lower on nilotinib 300 mg BID vs imatinib Cen, censored; EMR, early molecular response; Evt, events; Pts, patients. a OS rates reported consider each year to consist of twelve 28-day cycles. 37 Data cutoff: May 22, 2013

38 Progression to AP/BC on Study a (Including After Treatment Discontinuation) ENESTnd 5-Year Update P =.0588 P =.0047 New events in year % 3.5% 2.1% Imatinib 400 mg QD (n = 283) Nilotinib 300 mg BID (n = 282) Nilotinib 400 mg BID (n = 281) Two new progressions on study in year 5 (1 in the nilotinib 300 mg BID arm and 1 in the imatinib arm) Both patients had BCR-ABL > 10% at 3 months a Includes progression to AP/BC (excluding clonal evolution) or deaths in patients with advanced CML occurring on study (on core or extension treatment or during follow-up after treatment discontinuation). Data cutoff: May 22, 2013

39 PFS and OS on Study (Including After Treatment Discontinuation) a ENESTnd 5-Year Update Imatinib 400 mg QD (n = 283) Nilotinib 300 mg BID (n = 282) Nilotinib 400 mg BID (n = 281) Estimated 5-year PFS, % Progressions and deaths, n Hazard ratio (95% CI) 0.92 ( ) 0.46 ( ) P value Estimated 5-year OS, % Total deaths, n Deaths in patients with advanced CML, n b Hazard ratio (95% CI) 0.84 ( ) 0.46 ( ) P value There were 6 newly reported deaths in year 5 Imatinib (n = 2): both due to study indication Nilotinib 300 mg BID (n = 3): study indication, rectal cancer, and pneumonia Nilotinib 400 mg BID (n = 1): sepsis a Includes events occurring on core or extension treatment or during follow-up after treatment discontinuation. b Patients for whom the principle cause of death was either study indication or unknown or not reported but occurred subsequent to a documented progression to AP/BC. 39 Data cutoff: May 22, 2013

40 If we think that TKI therapy discontinuation is becoming our main goal, which is the best initial therapy?

41 To stop imatinib, it is necessary to achieve and sustain very low level of residual disease 6 months The median follow-up after discontinuation is 7.5 yrs (range: yr) Mean 7.1 yrs. At 5 years 50% (95% CI: 22-74) Cumulative incidence of molecular relapses Competing event = death in CMR without any relapse (n = 1) Michor F et al., Nature, 2005; 435: Rousselot et al., Blood, 2007;109:58 60.

42 ENESTnd 5-Year Update Cumulative Incidence of MR 4.5 Patients With MR 4.5, % Nilotinib 300 mg BID (n = 282) Nilotinib 400 mg BID (n = 281) Imatinib 400 mg QD (n = 283) Δ 6% to 10% By 1 Year a 11%, P < %, P < % By 4 Years a 40%, P < %, P =.0002 Δ 14% to 17% 23% By 5 Years a 54%, P < %, P <.0001 Δ 21% to 23% Time Since Randomization, Calendar Years 31% MR 4.5, molecular response 4.5-logs (BCR-ABL IS %). a Cumulative response rates reported consider each year to consist of twelve 28-day cycles. 42 Data cutoff: May 22, 2013

43 Proportion of Patients With MR 4.5 by BCR-ABL Levels at 3 Months ENESTnd 5-Year Update Nilotinib 300 mg BID Imatinib 400 mg QD BCR-ABL IS 1%: 56% of pts BCR-ABL IS 1%: 16% of pts Patients With MR 4.5, % BCR-ABL Level 1% > 1% to 10% > 10% Pts % P = % P = % MR 4.5 by 4 Years a MR 4.5 by 5 Years a 70% 52% 8% P =.0046 P = Time Since Randomization, Calendar Years Patients With MR 4.5, % BCR-ABL Level 1% > 1% to 10% > 10% Pts MR 4.5 by 4 Years a 65% P < % 5% P =.0001 MR 4.5 by 5 Years a 67% 34% 15% P =.0001 P = Time Since Randomization, Calendar Years Patients with BCR-ABL 1% at 3 months have significantly higher rates of MR 4.5 by 5 years More patients achieve BCR-ABL 1% at 3 months on nilotinib 300 mg BID vs imatinib a Cumulative response rates reported consider each year to consist of twelve 28-day cycles. 43 Data cutoff: May 22, 2013

44 Efficacy: Ø OS Ø MR4.5 Risk: ØLong term toxicity

45 Discontinuation is Possible The overall probability of maintenance of CMR at 24 and 36 months was 39% (95% CI 29-48). Molecular relapse occurred in 61 pts with 58 relapses occurring during the first 7 months 3 late relapses at month 19, 20 and 22, respectively Mahon FX, et al. Blood 2011;118:abstract 603

46 After discontinuation, some patients may show fluctuation in MRD CML patient in CP 31 years old Low risk Sokal score Rousselot P, personal data.

47 Kaplan-Meier estimate of TFR defined as loss of MMR and defined as loss of CMR 100 Percent survival % 37% P = Months Rousselot P et al., JCO in press TFR= treatment free remission

48 (i) (ii) (iii) (iv) (v) (vi) Goh et al. Leukemia &Lymphoma

49 49 Goh et al. Leukemia &Lymphoma 2011

50 Real Time PCR in CBFb-MYH11 positive AML patients CBFβ/MYH11 ABL X 10 4 copies R CCR pts. Relapsed pts R R R R R 25 R THRESHOLD LEVEL THRESHOLD LEVEL MONTHS Guerrasio et al., Leukemia 2002

51 Leukemia stem cells ØLSC could be entirely eradicated by imatinib therapy or by second generation TKI, at least in some patients but. ØIn some cases LSC seem to be resistant to TKI

52 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 cells BCR-ABL inhibition cells that survive and return to normality

53 Why CML stem cells survive TKI treatment? What are the possible differences between normal and CML stem cells? How can we dissect them?

54 Unknown genetic defects: Targetable differences? Known regulators of HSC maintenance Adapted from Akala et al. Curr Opin Genet Dev 2006, 16:

55 Next Generation Sequencing ER Mardis. Nature-2011;470:

56

57 CML patient SCs Ph- SCs NGS NGS?Exploitable Differences?

58 How Can Leukemic Stem Cells be Targeted? Targeting critical pathways for SC self renewal Hedgehog (LDE225) 1 Disrupting interaction of leukemic stem cells with the bone marrow stroma Janus kinase (JAK2) 2 CXCR4s blockers Using immunologic approaches Interferon-a 3 Vaccines Using other strategies in combination with nilotinib Autophagy inhibitors 1. Medina et al., Clin Transpl Oncol. 2009;11(4): Kuroda and Taniwaki. Curr Cancer Ther Rev. 2009;5: Essers et al., Nature. 2009;458(7240): Isaacs et al., Cancer Cell Mar;3(3): Botrugno et al., Cancer Lett. 2009;208:

59 CML - The Ph Model The multistep evolution, the profound knowledge (although not yet exhaustive) of the biology underlying the onset and the progression of the disease and the potential curability of the disease even using simple approaches of targeted therapy, make it probably the best model at our disposition to understand how to cure leukaemia

60 Grazie per l attenzione