Activating Mutations Observed in De Novo Acute Myeloid Leukemia Are Also Present in a Subset of Philadelphia Chromosome (Ph)-Positive Leukemia Patients with BCR-ABL1-Independent Resistance to ABL1 Kinase Inhibitors
Authors: Eide Christopher A. , Savage Samantha L. , Agarwal Anupriya , Bottomly Daniel , Wilmot Beth , McWeeney Shannon K. , Kurtz Stephen E. , O'Hare Thomas , Deininger Michael W. , Loriaux Marc M. , Tognon Cristina E. , Tyner Jeffrey W. , Druker Brian J. 
Published: October 2019

Abstract
The advent of ABL1 tyrosine kinase inhibitors has radically transformed the treatment and outcomes for patients with chronic myeloid leukemia (CML). However, 15-20% of newly diagnosed chronic phase patients demonstrate resistance to imatinib by 5 years, and responses are virtually always transient for patients with blast crisis CML or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). Acquisition of point mutations within the BCR-ABL1 kinase domain which compromise drug binding is the most common and well-studied cause of resistance to imatinib, and such mutations are generally well-handled by the newer, more potent ABL1 inhibitors nilotinib, dasatinib, and ponatinib. In the remainder of patients with resistance, however, sustained inhibition of BCR-ABL1 kinase activity is no longer sufficient to inhibit cell growth, implicating activation of additional co-critical BCR-ABL1-independent mechanisms of growth and survival. To identify and validate novel pathways important in BCR-ABL-independent resistance to ABL kinase inhibitors, we screened a cohort of 63 patients with CML (n=53) or Ph+ ALL (n=10) exhibiting clinical resistance to at least one ABL1 kinase inhibitor without an explanatory BCR-ABL1 kinase domain mutation. Following informed consent, primary mononuclear cells were isolated by Ficoll gradient centrifugation. Genomic DNA was isolated and subjected to whole exome sequencing on an Illumina HiSeq instrument with an average coverage depth of 30X. For samples with sufficient material, cells were also plated ex vivo in the presence of a panel of clinical and pre-clinical small-molecule kinase inhibitors, and assessed for effects on viability after 3 days. Among considerable heterogeneity of detected sequence variants, we observed a subset of patients who harbored mutations in genes reported to be frequently mutated in de novo acute myeloid leukemia (AML) (The Cancer Genome Atlas Research Network, NEJM 2013). Specifically, we observed recurrent mutations in FLT3 (n=3), DNMT3A (n=2), EZH2 (n= 6), IDH1 (n=4), KIT (n=4), KRAS (n=2), PTPN11 (n=2), RUNX1 (n=2), TET2 (n=5), and TP53 (n=2). These mutations were predominantly detected in patients with blast crisis CML, consistent with previous gene expression studies implicating common pathways involved in both resistance and progression (Radich et al., PNAS 2006; McWeeney et al., Blood 2010). Results from accompanying inhibitor revealed 1) ex vivo resistance to ABL1 kinase inhibitors largely tracked with clinical resistance profiles and 2) considerable variation in sensitivity profiles for other inhibitors, though samples harboring mutations in the genes above were associated with sensitivity to relevant kinase inhibitors. For one example, a CML lymphoid blast crisis patient who had previously failed imatinib, dasatinib, and nilotinib had a FLT3 D835Y mutation detected at the time of subsequent failure to ponatinib. Notably, while ponatinib inhibits native and certain mutated forms of FLT3, mutations of the D835 residue confer resistance to this drug (Smith et al., Blood 2013). This patient’s CML cells demonstrated significant ex vivo sensitivity to the FLT3 inhibitors tandutinib and crenolanib, whose sensitivity profiles include D835 variants. Taken together, our findings suggest that the molecular underpinnings of a subset of patients with Ph+ leukemia who become refractory to ABL1 kinase inhibitors without a BCR-ABL1 kinase domain mutation to explain their resistance may involve similar activating mutations to those observed in AML. These results warrant further investigation of inhibitors of involved pathways alone and in combination with ABL1 kinase inhibitors as a molecularly targeted therapeutic strategy in such patients.

Disclosures
Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy. Tyner:Constellation Pharmaceuticals: Research Funding. Druker:Astrazeneca: Consultancy; CTI Biopharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Scientific Founder, Scientific Founder Other; Cylene Pharmaceuticals: Consultancy, Equity Ownership; D3: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Lorus Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Stock options Other; MolecularMD: Consultancy, Equity Ownership, Scientific Founder. Some clinical trials on which I participate as PI or co-investigator utilize MolecularMD for molecular testing. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU., Scientific Founder. Some clinical trials on which I participate as PI or co-investigator utilize MolecularMD for molecular testing. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. Other; Novartis: Clinical trial funding: PI and co-investigator on Novartis clinical trials. OHSU has contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. , Clinical trial funding: PI and co-investigator on Novartis clinical trials. OHSU has contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Other, Dr. Druker is the inventor of a technology that is licensed to Novartis. This financial conflict of interest has been reviewed and managed by OHSU. Patents & Royalties; ARIAD: Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Other; Bristol-Myers Squibb: Clinical trial funding: PI and co-investigator on BMS clinical trials. OHSU has contracts with BMS to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. , Clinical trial funding: PI and co-investigator on BMS clinical trials. OHSU has contracts with BMS to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Other; Millipore: Dr. Druker is the inventor of a technology that is licensed to Millipore. This financial conflict of interest has been reviewed and managed by OHSU Patents & Royalties; Misc other pharmaceutical companies: Dr. Druker is an inventor on OHSU patent #843, licensed to many companies including but not limited to ARIAD; Array BioPharma; Curis; MolecularMD; Pfizer; Piramal Health Care; Praecis; SGX; The Translational Genomics Research Institute; and Vertex., Dr. Druker is an inventor on OHSU patent #843, licensed to many companies including but not limited to ARIAD; Array BioPharma; Curis; MolecularMD; Pfizer; Piramal Health Care; Praecis; SGX; The Translational Genomics Research Institute; and Vertex. Patents & Royalties.