Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.

BCR-ABL1-specific tyrosine kinase inhibitors prolong the life of patients with chronic myeloid leukemia (CML) but cannot completely eradicate CML progenitors. The BH3 mimetic, ABT-263, targets prosurvival BCL2 family members, and has activity against CML progenitors. However, the inhibitory effect of ABT-263 on BCL-XL, which mediates platelet survival, produces dose-limiting thrombocytopenia. A second-generation BH3 mimetic, ABT-199, has been developed to specifically bind BCL2 but not BCL-XL. We determined the activity of ABT-199 against CML cell lines, as well as primary CML and normal cord blood (NCB) progenitors. We find that BCL2 expression levels predict sensitivity to ABT-199 in CML and NCB progenitors, and that high NCB BCL2 levels may explain the reported hematologic toxicities in ABT-199-treated patients. Also, while single agent ABT-199 has modest activity against CML progenitors, when combined with imatinib, ABT-199 significantly enhances imatinib activity against CML progenitors at concentrations predicted to avoid hematologic toxicities.

Germline polymorphisms and tumor-specific genetic mutations together contribute to the behavior of human cancers, including the response to therapy. However, few specific models allow for the detailed study of how inherited and acquired genetic factors interact to cause clinical drug resistance, nor how their interaction can be prevented or overcome. We recently reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to targeted therapies in cancer, including the example of imatinib in chronic myeloid leukemia (CML) (Ng et al. Nature Medicine, 2012). Mechanistically, the deletion polymorphism led to the generation of BIM splice forms that lacked the pro-apoptotic BH3 domain, and that were incapable of activating apoptosis in response to targeted therapy. Clinically, we found that CML patients with the deletion polymorphism experienced inferior responses to imatinib. Furthermore, within the cohort of resistant patients harboring the BIM deletion polymorphism, we found that the majority developed cross-resistance to second or third generation TKI (nilotinib, dasatinib, and bosutinib), and that TKI resistance-conferring BCR-ABL kinase domain mutations were less frequent in patients with the BIM deletion polymorphism than those without. These observations suggested that the BIM deletion polymorphism cooperates with both BCR-ABL-independent and -dependent TKI resistance mechanisms to produce broad resistance to TKI. We generated K562 cells with and without the BIM deletion polymorphism that were resistant to imatinib by using the method described by Mahon et al (Blood, 2000). We found that deletion-containing cells exhibited 3- to 4-fold increased viability as compared to wildtype cells, and this was due to reduced apoptosis in the deletion-containing cells. We then compared the panel of wildtype and deletion-containing cells for the development of known TKI resistance mechanisms. The table below summarizes our findings. Importantly, we also found that, in a number of cells, both ERK and LYN activation occurred independently of BCR-ABL.Known TKI resistance mechanismWildtypeHeterozygous for BIM deletion polymorphismHomozygous for BIM deletion polymorphismSomatic mutation in BCR-ABL kinase domain--G250EBCR-ABL gene amplification--+++LYN activation+++++ERK activation+++-++ Next, we tested the ability of second-generation TKI, including dasatinib and nilotinib, to overcome resistance induced by long-term culture. We also determined if the addition of ABT-737, a BH3-mimetic, to the TKI could overcome resistance in our cell lines. First, we found that prolonged incubation with imatinib induced cross-resistance to both nilotinib and dasatinib, a result that reproduced our observation in patients with the BIM deletion polymorphism. Next, we found that a combination of second generation TKI, especially dasatinib, and ABT-737 induced maximal cell death in cells with the BIM deletion polymorphism. Based on the information generated from our cell lines, we evaluated the viability of TKI-resistant primary CML cells when treated with TKI with and without ABT-737. In TKI-resistant primary CML cells, regardless of their BIM deletion status, maximal reduction in cell viability was observed when both TKI and ABT-737 were used. We conclude that: 1. The BIM deletion polymorphism is permissive for the acquisition of somatic events that mediate both BCR-ABL-dependent and -independent mechanisms of TKI resistance. 2. The permissive action of the BIM deletion polymorphism is associated with reduced apoptosis, and likely accounts for the ability of BIM deletion polymorphism-containing CML cells to acquire secondary resistance-conferring events. 3. The combined use of TKI with ABT-737 represents a potential therapeutic strategy to overcome TKI resistance in CML patients with and without the BIM deletion polymorphism. 4. In our cell line system, dasatinib with ABT-737 induced maximum cell death. Finally, our results demonstrate that germline and acquired resistance factors can interact to produce resistance to targeted therapies in cancer, and that cell line systems allow us to both elucidate these mechanisms as well as devise therapeutic strategies to overcome such resistance. Disclosures: Chuah: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Both germline polymorphisms and tumor-specific genetic alterations can determine the response of a cancer to a given therapy. We previously reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), as well as other cancers [1]. The deletion polymorphism favored the generation of BIM splice forms lacking the pro-apoptotic BH3 domain, conferring a relative resistance to the TKI imatinib (IM). However, CML patients with the BIM deletion polymorphism developed both partial and complete IM resistance. To understand the mechanisms underlying the latter, we grew CML cells either with or without the BIM deletion polymorphism in increasing IM concentrations. Under these conditions, the BIM deletion polymorphism enhanced the emergence of populations with complete IM resistance, mimicking the situation in patients. Importantly, the combined use of TKIs with the BH3 mimetic ABT-737 overcame the BCR-ABL1-dependent and -independent resistance mechanisms found in these cells. Our results illustrate the interplay between germline and acquired genetic factors in confering TKI resistance, and suggest a therapeutic strategy for patients with complete TKI resistance associated with the BIM deletion polymorphism.