A Common Deletion Polymorphism in the BIM Gene Contributes to Intrinsic Imatinib Resistance in Chronic Myelogenous Leukemia

Abstract 1666

The use of the tyrosine kinase inhibitor (TKI), imatinib (IM), to target the oncogenic BCR-ABL kinase has resulted in profound responses in patients with chronic phase (CP) chronic myelogenous leukemia (CML). However, a subset of patients do not respond to TKIs, and are deemed to have primary resistance. Importantly, patients with European LeukemiaNet (ELN)-defined ‘failure’ or ‘suboptimal response’ are at increased risk of poorer long-term outcomes. Little is known about mechanisms underlying primary resistance, where only a minority of patients have BCR-ABL kinase domain (KD) mutations. Interestingly, East-Asian CML patients are reported to have lower complete cytogenetic response rates compared to the West (∼50 vs 80% respectively, Au et al. 2009). We used massively parallel DNA sequencing of paired-end ditags to identify genetic factors associated with resistance in CML patient samples. We discovered a novel deletion polymorphism in the BIM gene that correlated with resistance, and which represented a common polymorphism in normal East-Asian (12.3% carriers), but not African or Caucasian (0%), populations (n=2465). BIM is a pro-apoptotic BCL2 family member, and plays a central role in CML pathophysiology. Here, BCR-ABL suppresses FoxO3a-mediated BIM transcription to maintain a survival advantage, while preventing BIM expression following BCR-ABL inhibition results in TKI resistance. Inspection of BIM gene structure suggested the polymorphism would result in mutually exclusive splicing of exon 3 (E3) vs 4 (E4), leading to decreased expression of BIM transcripts encoding the pro-apoptotic BH3 domain (found only in E4). To test this hypothesis, we constructed a minigene to measure E3 vs E4 splicing, and found the polymorphism decreased splicing to E4 over E3 by >5-fold. Importantly, primary CML cells exhibited the same phenomenon, since polymorphism-containing samples expressed lower levels of E4- vs E3-containing transcripts (p=0.008), while general BIM transcription was unaffected. Our observations suggested a novel mechanism for intrinsic TKI resistance. Here, upon IM exposure, polymorphism-containing CML cells would favor induction of E3- vs E4-containing BIM transcripts, decreased expression of BH3-containing BIM isoforms, and impaired apoptosis. To facilitate these studies, we identified a Japanese CML cell line, KCL22, which contained the polymorphism, and confirmed it had a decreased E4/E3 transcript ratio compared to cells without the polymorphism. KCL22 cells also had decreased induction of E4-containing transcripts following IM, as well as lower levels of BIMEL protein, a major BH3-containing BIM isoform. Consistent with prior reports, KCL22 cells were resistant to IM, despite effective BCR-ABL inhibition, and had impaired apoptotic signalling upon IM exposure. Importantly, and as predicted by our model, pharmacologic restoration of BH3 activity (using the BH3-mimetic drug, ABT-737) sensitized cells to IM-induced death. Next, we used zinc finger nuclease-facilitated gene targeting to precisely create the polymorphism in the BIM gene of IM-sensitive K562 CML cells. We generated subclones that were heterozygous or homozygous for the polymorphism, and confirmed a decreased E4/E3 ratio in these cells in a polymorphism-dosage-dependent manner. Polymorphism-containing cells exhibited decreased induction of E4-containing transcripts following IM exposure, as well as impaired upregulation of BIMEL protein, and diminished apoptotic cell death. As in KCL22 cells, ABT-737 enhanced the ability of IM to activate apoptosis in polymorphism-containing cells. Using an expanded East-Asian CML cohort (n=203), we found the polymorphism correlated with TKI resistance (defined as ‘failure’ or ‘suboptimal’ per ELN criteria) in CP patients treated with 400 mg IM daily (p=0.02). Further, patients with the polymorphism were more likely to have resistance in the absence of a KD mutation than those without (OR=2.24, 95% CI of 1.22–4.12). In sum, we have found an East-Asian polymorphism in BIM that is associated with intrinsic resistance to TKIs. Screening for this polymorphism may be useful in identifying patients at risk of TKI resistance; a resistance we show can be overcome by BH3 mimetics. Our findings may also apply to other cancers and proliferative disorders in which drug-sensitivity is BIM-dependent.