Imatinib Resistance Geoffrey L. Uy, M.D. Associate Professor of Medicine Division of Oncology

Natural History of CML Chronic PhaseAccelerated PhaseBlast Phase Duration3-5 yrs, untreatedVariesMedian of months PrognosisResponsiveLess responsiveResistant SymptomsAsymptomatic Fatigue Weight loss Abdominal pain or discomfort Night sweats Progressive Splenomegaly Marrow fibrosis Bleeding Infections Time

Disease Monitoring in CML Hematologic response – Complete normalization of peripheral blood counts – No immature cells in peripheral blood (ie. Blasts) – No signs or symptoms of disease such as splenomegaly Sufficient when therapy was relatively ineffective

Disease monitoring in CML (Conventional Cytogenetics) Grow cells in culture and arrest in metaphase with colchicine Chromosomal banding Giemsa-banding G-banding regions AT-rich and gene poor. Analyze cells (~5% sensitivity) Good for large gains, losses, & translocations Not sensitive for small lesions

Disease monitoring in CML (Fluorescent in-situ Hybridization, FISH) Dual color, dual fusion probe BCR-ABL BCR Abl ABL-BCR Sensitivity 1:200-1:500 Can detect very small gains, losses and translocations Need to know what you are looking for

Disease monitoring in CML (qRT-PCR) Make a cDNA library from the mRNA specimen Design quantititive real time PCR to amplify across different fusion breakpoints Detect specific transcript based on use of allele specific probe Quantify relative expression to control gene, (B2-microglobulin or Abl) Detect 1 in 10 6 Bcr-Abl transcripts

Hematologic H&E stain Peripheral Blood (with myeloid cells) Bone Marrow (myeloid hyperplasia) Karyotype (Ph chromosome) Abnormal BCR-ABL Red= BCR Green= ABL Yellow=Fusion FISH Chromosomal translocation t(9;22)(q34;q11) Cytogenetic Molecular (BCR-ABL fusion) PCR Abnormal BCR-ABL Lane 1= BCR-ABL+ sample Lane 2= BCR-ABL- sample Monitoring CML Sensitivity

Imatinib in CML Druker et al., N Engl J Med Dec 7;355(23): Cumulative Best Response to Initial Imatinib Therapy.

Outcome of Imatinib by Response Druker et al., N Engl J Med Dec 7;355(23):

Therapeutic Landmarks Months of treatment Treatment FailureSuboptimal response 3No Hematologic Response Less than Complete Hematologic Response 6 Ph+ cells >35 percent 12Ph+ cells >35 percent Less than complete Cytogenetic Response 18 Less than complete Cytogenetic Response Less than major Molecular Response

Clinical Observations with Imatinib Virtually all patients with chronic phase disease respond to Imatinib (but to different degrees) Patients with advanced disease / blast crisis have very transient responses When imatinib is stopped, disease returns, ie. imatinib is NOT a cure

Why doesn’t Imatinib work in everyone? Drug Intolerance – Inability of a patient to continue therapy despite optimal management of side effects Primary Resistance (Treatment-refractory Ph+) – Lack of efficacy after treatment initiation despite use of therapy at appropriate doses Secondary Resistance (Acquired resistance) – Loss of efficacy despite use of therapy at optimal doses

Is Imatinib Resistance BCR-ABL Dependent? Expose bone marrow from patient with CML to increasing amounts of imatinib Western blot for BCR- ABL, CRKL and P-CRKL Gorre et al. Science 2001 Aug 3;293(5531):

BCR-ABL Activity in Relapsed Patients Take samples from patients with relapsed disease and look for P-CRKL Examine samples before, during treatment and at relapse Imatinib resistance associated with reappearance of functional BCR-ABL ie. BCR-ABL dependent Gorre et al. Science 2001 Aug 3;293(5531):

How does this happen???

BCR-ABL FISH in Relapsed Patients (A)Patient with blast crisis during imatinib therapy (B)2 nd patient before, during and after therapy (C)FISH from patient showing duplicated inverted Ph- chromosome Gorre et al. Science 2001 Aug 3;293(5531):

Sequencing of ABL Kinase Domain Gorre et al. Science 2001 Aug 3;293(5531):

Model of Imatinib Binding of ABL Gorre et al. Science 2001 Aug 3;293(5531):

BCR-ABL Kinase Domain Mutations

Ba/F3 Model of CML growth BCR-ABL CMV + IL-3 - IL-3 Ba/F3 - IL-3 + Imatinib

Ba/F3 Model of CML growth BCR-ABL(T315I) CMV Ba/F3 - IL-3 + Imatinib

Mechanisms of Imatinib Resistance Gene amplification ABL kinase domain mutations Other mechanisms observed in vitro only – activation of BCR-ABL–independent pathways (Src family proteins) – increased drug efflux through the multidrug resistance gene

Overcoming Imatinib resistance?

Overcoming Imatinib Resistance Novel BCR-ABL inhibitor, dasatinib (aka. BMS ) in Ba/F3

Dasatinib against mutant BCR-ABL in Ba/F3 cell assays Most BCR-ABL mutations except T315I are sensitive to dasatinib

Dasatinib in mouse model of CML BCR-ABL luciferase transduced Ba/F3 cells Injected into SCID mice Treat with Dasatinib or vehicle Shah et al., Science 305, 399 (2004);

Dasatinib in Imatinib-Resistant Philadelphia Chromosome-Positive Leukemias Talpaz et al., N Engl J Med (24):

Sensitivity of Bcr-Abl Mutations O'Hare T et al. Blood 2007;110:

Clinical Responses to Dasatinib According to Mutation Type

Targeting T315I - Ponatinib Rationally designed inhibitor of BCR-ABL Active against T315I mutant – Unique approach to accommodating gatekeeper residue – Binds inactive (closed) ABL conformation Ponatinib cocrystal structure with ABL T315I Ponatinib

Ponatinib in Ba/F3 Assay (*P < 0.05) *** (*P < 0.01) AB O’Hare T, et al. Cancer Cell. 2009;16:

Conclusions Both primary & secondary resistance is rare in chronic phase CML but extremely common in advanced stage, blast crisis CML Mutations in Abl kinase domain are dominant mechanism for disease resistance Understanding mechanisms of resistance can lead to rapid development of novel agents to overcome resistance

Approach to Treatment of CML Routine monitoring of patient cytogenetics, FISH and RT-PCR for BCR-ABL If patients fail to meet therapeutic landmarks Evaluate for kinase domain mutations – Increase dose of imatinib – Change to dasatinib or nilotinib – Clinical trial (3 rd generation inhibitors) – Allogeneic stem cell transplant