49 Reversal of Mutant KRAS-Mediated Apoptosis Resistance by Concurrent Bcl-xL Antagonism and Induction of Pro-Apoptotic BH3-Only Proteins in Colorectal Carcinoma Cells

Abstract

Background. KRAS mutations, detected in at least 40% of human colorectal cancers (CRC), contribute to de novo apoptosis resistance and ultimately treatment failure. Aims. To study the mechanism of KRAS-mediated apoptosis resistance, we examined proand anti-apoptotic Bcl-2 family proteins in isogenic mutant vs wild type KRAS colorectal cancer (CRC) cells. Experimental Methods. Isogenic KRAS mutant CRC cells (HCT116, DLD1) and KRAS mutant SW620 cells were analyzed for Bcl-2 family proteins by immunoblotting. Cells were incubated with ABT-263 (1 μM), carfilzomib (0-50nM) or their combination; apoptosis was measured by annexin V+ labeling and caspase cleavage. Drug synergy was determined by calculation of a combination index. Protein-protein interaction was examined by immunoprecipitation. Knockdown of ERK or KRAS was achieved by siRNA. Ectopic expression of mutant KRAS or Noxa was achieved by a constitutive or doxycyline-inducible expression system, respectively. Results. We found that mutant KRAS cells showed upregulation of Bcl-xL that was attenuated by KRAS siRNA or reproduced by ectopic expression of mutant KRAS. Upregulation of Bcl-xL by mutant KRAS was mediated by ERK shown using EKR siRNA. Bcl-xL upregulation occurred at the level of mRNA and protein shown using actinomycin D and cyclohexamide, respectively. While BH3 mimetic agents can antagonize Bcl-2/BclxL, they are generally insufficient to overcome apoptosis resistance in solid tumors. Therefore, we evaluated a strategy of concurrent Bcl-xL antagonism and induction of pro-apoptotic BH3-only proteins. Carfilzomib, an irreversible proteasome inhibitor, was shown to induce Noxa and Bik proteins but failed to reverse mutant KRAS-mediated apoptosis resistance. However, concurrent antagonism of Bcl-xL by the BH3 mimetic, ABT-263, combined with carfilzomib was shown to synergistically enhance apoptosis in isogenic mutant KRAS CRC cell lines (Figure). In support of this strategy, we mimicked Noxa induction, seen with carfilzomib, using a doxycycline-inducible expression system. Ectopic Noxa was shown to markedly enhance apoptosis by ABT-263. A mechanism for drug synergy was suggested by the finding that Noxa and Bik proteins can sequester anti-apoptotic Mcl-1, while ABT-263 was able to release Bik and Bak from binding to Bcl-xL. Conclusion. Our novel findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance. Antagonism of Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. Together, these preclinical data suggest a promising therapeutic strategy to overcome apoptosis resistance in KRAS mutant CRC cells.