BIM is a pro-apoptotic member of the BCL-2 family of proteins that trigger apoptosis by either opposing the pro-survival members of the family (BCL-2, BCL-xL, MCL1), or by directly binding the distal apoptotic effectors, BAX and BAK. Importantly, BIM expression is required for sensitivity towards tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) and EGFR-mutated non-small cell lung cancer (EGFR NSCLC) cell lines, since BIM knockdown in these cells results in TKI resistance. Using paired-end DNA sequencing, we recently discovered a novel 2.9kb deletion polymorphism in intron 2 of the BIM gene that was associated with TKI resistance in CML and EGFR NSCLC cell lines. When present, the deletion polymorphism results in the preferential expression of exon 3 (E3)- vs exon 4 (E4)-containing BIM isoforms in a mutually exclusive manner, since E3 also contains a stop codon and polyadenylation signal sequence. As a result, expression of E4-containing BIM isoforms (e.g. BIMEL, BIML, BIMS) that lack the pro-apoptotic BH3 domain is impaired. This then leads to defective apoptotic signaling in the presence of TKIs, and intrinsic TKI resistance. In this study, we aimed to identify cis-regulatory elements within the deleted fragment that suppress splicing to E3, and to assess the apoptotic activity of the major E3-containing isoform, BIMγ. Using serial deletion analysis, we have mapped the cis-regulatory elements to a 150-nucleotide (nt) region within the deletion. Furthermore, our results also showed that the deletion does not create a novel element that regulates E3 splicing. Ongoing analysis is expected to narrow the region to the 6-7 nt usually required for splicing factor binding, as well as to aid in identifying the relevant splicing factors. Next, we assessed the apoptotic activity of BIMγ, and found that the BIMγ protein was markedly less stable (∼T1/2 <1hr) than E4-containing BIM isoforms (BIML) (∼T1/2 ∼4hrs). Second, forced expression of BIMγ failed to affect cell viability, in contrast to E4-containing isoforms. Lastly, knockdown of BIMγ failed to sensitize a TKI resistant CML cell line harboring the deletion polymorphism (KCL22) to TKI-induced apoptosis. In summary, we have mapped cis-regulatory elements suppressing the splicing of E3 to a 150 nt region within the deletion. We also show that the BIM deletion polymorphism favors the generation of unstable E3-containing BIM isoforms, which, even when overexpressed, lack the pro-apoptotic activity of E4-containing BIM isoforms. Together, these data provide a mechanism by which the deletion polymorphism mediates TKI resistance in CML and EGFR NSCLC cell lines. Importantly, our results identify potential targets for modulating alternative splicing of BIM as a therapeutic approach for overcoming TKI resistance mediated by the BIM deletion polymorphism.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4958. doi:1538-7445.AM2012-4958