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Abstract 3504 The imatinib paradigm established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKIs). However, once-daily dasatinib (serum half-life: 3–5 h) is clinically effective despite only transient BCR-ABL inhibition, opening an opportunity for in-depth study of the mechanistic requirements for ABL TKI-induced CML cell death. Apoptosis commitment after potent, transient target inhibition is observed with ABL TKIs in vitro (Blood, 114, 2009, 3459–63), although variations in required TKI concentrations relative to their activity against BCR-ABL kinase suggest involvement of previously unrecognized factors. The “oncogenic shock” concept holds that temporary disruption of BCR-ABL-mediated prosurvival and proapoptotic signaling sets up a kinetic imbalance in favor of apoptosis. We have undertaken a comprehensive mechanistic exploration of this issue, wherein CML cells were transiently exposed to the ABL TKIs imatinib (50 and 500 nM), nilotinib (50 and 500 nM), dasatinib (10 and 100 nM), and ponatinib (AP24534; 10 and 100 nM) and then investigated with respect to pathways critical to drug efficacy and intracellular residence time. Cellular studies utilized multi-parameter intracellular FACS and immunoblot analysis, liquid chromatography-mass spectrometry, and high-throughput real-time qPCR expression analysis. Corresponding biochemical studies to determine ABL kinase/inhibitor dissociation parameters were also performed. All four ABL TKIs tested were capable of triggering apoptosis following transient exposure, although nilotinib and imatinib (which feature much narrower kinase target profiles than dasatinib and ponatinib) did so only at high concentrations. In contrast to potent, transient inhibition of BCR-ABL being necessary and sufficient for commitment of CML cells to apoptosis, we found that apoptosis could be reversed under conditions involving extensive additional TKI washout protocols. Consistent with the best indicator of apoptosis induction in our experiments being incomplete restoration of BCR-ABL signaling activity to pre-treatment levels, in all cases for which apoptosis commitment was irreversible, we identified a small, functionally important pool of intracellular TKI after washout of drug from the media. This property correlated with results of ABL kinase/inhibitor dissociation studies. For example, we found that ponatinib is a tight-binding inhibitor with a remarkably slow off-rate (t1/2 >95 h). Transient exposure to ponatinib followed by thorough washout committed CML cells to apoptosis despite very low intracellular concentrations that did not completely inhibit BCR-ABL. Based on these findings, we explored the possibility that effective TKIs are inhibiting additional targets involved in committing cells to an apoptotic fate, and used high-throughput qPCR assays to identify a preliminary profile of 30 apoptosis-related genes differentially expressed in TKI-treatment conditions that do and do not irrevocably commit CML cells to apoptosis. Taken together, our findings reveal that even slightly attenuated restoration of BCR-ABL signaling correlates with apoptosis commitment and that cryptic cellular retention of ABL TKIs is important in mediating this effect, potentially via sustained low-level inhibition of auxiliary targets. By extension, monitoring intracellular drug levels by LC-MS may be informative, especially for short serum half-life kinase inhibitors such as dasatinib. These studies further establish and refine the guiding principles of commitment of CML cells to apoptosis and improve our ability to design kinase inhibitors for CML and other malignancies. Disclosures: Riddle: Life Technologies Corporation: Employment, Equity Ownership. Apgar:BD Biosciences: Employment. Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding. Druker:Bristol-Myers-Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential COI has been reviewed and managed by the OHSU COI in Research Committee & Integrity Program Oversight Council.

Abstract 3744 Background: BCR-ABL kinase domain (KD) mutations are a common mechanism of chronic myeloid leukemia (CML) resistance to tyrosine kinase inhibitor (TKI) therapy. It is well known that some patients harbor more than one KD mutation in the same sample, but the frequency of true compound mutations (defined as two or more mutations in the same allele) and the clonal relationships between mutant clones have not been established. Methods: The first group of samples (Group 1) was selected based on evidence of more than one BCR-ABL KD mutation by Sanger sequencing. Samples from a second group of patients (Group 2) who had one mutation in BCR-ABL KD by Sanger sequencing were also analyzed. The BCR-ABL KD was amplified using nested RT-PCR, the final amplicon was shotgun-cloned and 10 clones from each sample were sequenced. Results: Thus far, 18 samples from group 1 have been analyzed. Sequencing of 180 colonies from this group revealed >80 different mutations. Compound mutations were confirmed in 13 samples (Table 1), while 5 samples revealed the two mutations were in separate clones. T315I, F359V, V299L and M351T were over-represented among compound mutations and the T315I/F359V and V299L/M351T compound mutations were each detected in samples from two patients. On average, each patient sample had 7 different mutations per 10 clones (range: 3–14), some were silent or not previously shown to be associated with TKI resistance in biochemical or cell-based assays. In the 13 samples from Group 1 with confirmed compound mutations (Table 1), all respective individual mutations had been shown to confer TKI resistance in biochemical and/or in vivo studies. In 4 patients, the compound mutant clone was non-dominant (≤40% of clones). In 3 of these 4 patients (11, 13, 16; Table 1), both mutations were also detected independently in co-existent clones, while 1 patient (18) showed 2 compound mutant clones (T315/F359V and V299L/M351T) at comparable levels (40% each). In the remaining 9 patients from Group 1, the compound mutant represented >70% of clones. More than 2 mutations were observed in 41/102 screened compound mutant clones from Group 1. The number of mutations in these clones ranged from 3 to 6. Except V299L/M351T/E329G, which was observed in 5/10 screened clones of one patient (1), no clone with more than 2 mutations was detected in more than 1 clone per sample (≤10%). Notably, none of these additional mutations has yet been associated with TKI resistance in biochemical or cell-based assays. So far, samples from 7 patients in Group 2 have been analyzed. Sequencing of the clones from these patients also revealed the presence of low-level compound mutants. On average 30% of the clones had compound mutations (range: 20%-40%) and an average of 5 different mutations were detected among the 10 sequenced clones per patient (range: 3–6), some of which were silent or not previously shown to be associated with TKI resistance in biochemical or cell-based assays. Conclusions: 1. True compound mutations are common in patients with evidence of multiple mutations by direct sequencing. 2. Mutant clones evolve sequentially as well as in parallel, suggesting complex clonal relationships, in which the identical phenotype (mutation) may be acquired independently by multiple clones. 3. Clones harboring compound mutations comprised of more than 2 mutations rarely achieve dominance, suggesting that the number of different mutations compatible with maintenance of BCR-ABL kinase activity may ultimately be limited. 4. Low-level compound mutations are also detected by cloning and sequencing in the samples from patients who have had one detected mutation in screening (Group 2). Disclosures: Lange: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 1416 Among patients with chronic myeloid leukemia (CML) who develop resistance to ABL tyrosine kinase inhibitor (TKI) therapy, the most common explanation is acquisition of point mutations within the BCR-ABL kinase domain that interfere with drug binding. The second-generation ABL TKIs nilotinib and dasatinib have proven effective against most imatinib-resistant mutants, with the critical exception of BCR-ABLT315I. Furthermore, sequential treatment with ABL TKIs can select for BCR-ABL compound mutations (two or more point mutations within the same BCR-ABL molecule) that are resistant to multiple inhibitors. With third-generation ABLT315I TKIs such as ponatinib (AP24534) and DCC-2036 currently in clinical development, containment of single point mutation-based resistance appears tenable. However, the ability of available inhibitors to address resistance due to the growing number of reported BCR-ABL compound mutations has not been investigated. Here, we generated stable Ba/F3 cell lines expressing a panel of clinically-observed BCR-ABL compound mutants (including two novel mutants which we identified from CML clinical resistance specimens, BCR-ABLG250E/V299L and BCR-ABLT315I/H396R) as well as cells expressing each constituent mutation. Cells were plated in graded concentrations of imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036, incubated for 72 hours, and cellular proliferation was analyzed by standard methanethiosulfonate-based assay. We found that, consistent with our previous cell-based mutagenesis screens for resistance (Blood 2006, 108: 2332–8; Cancer Cell 2009, 16: 401–12; Cancer Research 2011, 71: 3189–95), all five tested inhibitors demonstrated unique but partially overlapping resistance profiles relative to the panel of compound mutants tested. As expected, all mutants harboring a T315I component were insensitive to imatinib, nilotinib, and dasatinib, whereas ponatinib and DCC-2036 showed varying levels of efficacy against such mutants. The most resistant, clinically reported compound mutants were BCR-ABLG250E/T315I, BCR-ABLE255K/T315I, and BCR-ABLE255V/T315I, all of which conferred markedly increased resistance to ponatinib and DCC-2036 relative to cells expressing each constituent mutation alone (Table 1). For example, the most resistant compound mutant, BCR-ABLE255V/T315I, conferred high-level resistance to ponatinib and DCC-2036 (IC50: 425 and 1272 nM, respectively), while cells expressing either BCR-ABLE255V or BCR-ABLT315I were inhibited at clinically relevant concentrations of ponatinib (IC50: 33 and 18 nM, respectively) or DCC-2036 (IC50: 659 and 149 nM, respectively). Additional structural, biochemical, and signaling pathway analyses addressing the striking differences in sensitivity of BCR-ABL compound mutants versus their constituent mutations are being pursued and will be presented. Overall, our findings demonstrate that BCR-ABL compound mutations confer varying degrees of resistance to currently available ABL TKIs, necessitating rational treatment selection to optimize outcomes. Patients harboring highly multi-drug-resistant compound mutants such as BCR-ABLE255V/T315I may have limited therapeutic options available, warranting investigation into combination therapies involving ABLT315I TKIs. Furthermore, studies of compound mutation-based resistance mechanisms in CML advance the possibility of maximum disease control in a greater proportion of patients and have important implications for anticipating resistance and designing treatment strategies in similar, more rapid clinical resistance scenarios such as those encountered with EGFR or ALK mutations in non-small-cell lung cancer.Table 1.Cell proliferation IC 50s for clinically-observed BCR-ABL compound mutants.Cell lineimatinib (nM)nilotinib (nM)dasatinib (nM)Ponatinib (nM)DCC-2036 (nM)Parental>5120>5120>768>768>5120Native BCR-ABL270121.22.337BCR-ABLG250E26391702.87.7180BCR-ABLG250E/T315I>5120>5120>76849627BCR-ABLE255K>51203268.923410BCR-ABLE255K/T315I>5120>5120>768106585BCR-ABLE255V>512011771333659BCR-ABLE255V/T315I>5120>5120>7684251272BCR-ABLT315I>5120>5120>76818149 Disclosures: Druker: Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Bristol-Myers-Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential COI has been reviewed and managed by the OHSU COI in Research Committee & Integrity Program Oversight Council.