Citations

Abstract Background. Oncogenic KRAS mutations occur in nearly one-half of colorectal cancers (CRC) where they contribute to de novo apoptosis resistance and treatment failure. Inhibition of MEK/ERK is a promising strategy to sensitize KRAS mutant tumors to apoptosis that may involve upregulation of pro-apoptotic BH3-only proteins, including BIM. However, BH3-only proteins can be neutralized by anti-apoptotic Bcl-xL that we found was upregulated in isogenic KRAS mutant compared to wild-type HCT116 and DLD1 CRC cells. Aims. We determined the ability and mechanism by which a novel MEK/ERK inhibitor, GDC-0623, can induce BIM to promote apoptotic signaling. A strategy of concurrent induction of BH3-only proteins and antagonism of Bcl-xL was studied to overcome mutant KRAS-mediated apoptosis resistance. Results. Using isogenic KRAS HCT116 cells and KRAS mutant SW620 cells, we found that GDC-0623 can potently induce the BH3-only protein BIM, and to a lesser extent BIK, in both KRAS wild-type and mutant cells. Only KRAS mutant cells remained resistant to GDC-0623-induced apoptosis. Knockdown of Bcl-xL or use of a BH3 mimetic drug, ABT-263, sensitized KRAS mutant cells to GDC-0623-mediated apoptosis. GDC-0623 and ABT-263 were shown to interact synergistically to promote apoptosis in KRAS mutant cells. Dependence on BIM or BIK for synergy between GDC-0623 and ABT-263 was shown by shRNA knockdown that each attenuated drug-induced apoptosis. Mechanistically, ABT-263 treatment was shown to release BIM from its binding to Bcl-xL using immunoprecipitation. The ability of GDC-0623 to induce apoptosis occurred via BIM that was phosphorylated by MEK/ERK activation leading to BIM degradation. GDC-0623 was shown to dephosphorylate BIM at serine 69 in cells with wild-type BIM or a phospho-mimic mutation (S69E). However, GDC-0623's ability to dephosphorylate BIM was lost in cells containing a phosphorylation-defective BIM mutation (S69G) that showed delayed BIM degradation. Conclusion. GDC-0623 was shown to dephosphorylate BIM to enhance its accumulation and to promote apoptosis. Concurrent induction of BIM/BIK and antagonism of Bcl-xL can overcome mutant KRAS-mediated apoptosis resistance, suggesting a promising therapeutic strategy against KRAS mutant CRCs. Note: This abstract was not presented at the meeting. Citation Format: Aziz Zaanan, Koichi Okamoto, Hisato Kawakami, Shengbing Huang, Frank Sinicrope. MEK/ERK inhibitor GDC-0623 dephosphorylates and accumulates BIM that enables a synergistic apoptosis with the Bcl-xL antagonist, ABT-263, in mutant KRAS colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2937. doi:10.1158/1538-7445.AM2015-2937

Abstract KRAS mutations are frequently detected in human colorectal cancer and contribute to de novo apoptosis resistance and ultimately therapeutic failure. To overcome KRAS-mediated apoptosis resistance, the irreversible proteasome inhibitor, carfilzomib, was evaluated and found to potently induce Noxa, which was dependent upon c-Myc, and Bik. Isogenic mutant versus wild-type KRAS carcinoma cells showed elevated Bcl-xL, confirmed by KRAS siRNA or ectopic expression. Upregulated Bcl-xL by mutant KRAS was mediated by ERK as indicated by ERK knockdown. Bcl-xL expression was regulated at the level of mRNA and protein as shown using actinomycin D and cyclohexamide, respectively. Suppression of Bcl-xL by shRNA sensitized mutant KRAS cells to carfilzomib. Concurrent Bcl-xL antagonism by the BH3 mimetic ABT-263 combined with carfilzomib synergistically enhanced apoptosis that was dependent on Bax or p53, and was attenuated by Noxa or Bik shRNA. In support of this strategy, ectopically expressed Noxa enhanced apoptosis by ABT-263. Carfilzomib-induced Noxa and Bik sequestered Mcl-1 and ABT-263 released Bik and Bak from Bcl-xL, suggesting a mechanism for drug synergy. These preclinical findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance in KRAS-mutant colorectal cancer. Furthermore, antagonizing Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. Implications: This novel study reveals a promising treatment strategy to overcome apoptosis resistance in KRAS-mutant colorectal cancer by concurrent upregulation of Noxa/Bik and antagonism of Bcl-xL. Mol Cancer Res; 13(4); 659–69. ©2014 AACR.

Key Points High-throughput RNAi screening identified lenalidomide sensitizer genes, including RSK2, RAB, peroxisome, and potassium channel family members. Knockdown or inhibition of RSK2 synergized with lenalidomide to induce myeloma cytotoxicity and downregulation of interferon regulatory factor 4 and MYC.