Abstract C72: Combined pan-RAF and MEK inhibition overcomes multiple resistance mechanisms to selective RAF inhibitors

Abstract

Abstract Background RAF and MEK inhibitors are efficacious in BRAF mutant melanoma but not in BRAF mutant colorectal cancer (CRC). To understand the underlying mechanisms of this difference we performed an RNA interference screen to identify loss of function events that sensitize CRC cells to RAF inhibition by PLX4720. Methods A pooled lentiviral library encoding 90,000 shRNAs, targeting 16,500 genes was transduced into the PLX4720-resistant, BRAF mutant RKO CRC cell line. The shRNA-infected cells were split into two experimental arms treated with either PLX4720 or DMSO. After 16 population doublings, the abundance of each shRNA was determined by PCR amplification and deep sequencing of the barcoded shRNA pool. The log-fold change in shRNA reads in the drug-treated condition was calculated relative to the vehicle control. RNAi gene enrichment (RIGER) was employed to rank genes that promoted sensitivity to PLX4720. Candidate genes were validated individually and their effects on cell proliferation, survival and MAPK pathway inhibition assessed. Results Genes involved in maintaining MAPK pathway activity scored prominently in our screen. Knockdown of MET, SHP2, SHOC2 and CRAF by shRNA sensitized cells to PLX4720. Efforts to suppress cell proliferation via single agent pan-RAF or MEK inhibition were more effective across melanoma and colorectal cancer cell lines than PLX4720. Strikingly, combined pan-RAF and MEK inhibition elicited a synergistic response in cell lines showing intrinsic resistance to PLX4720 associated with NF1 or KRAS mutation. Conclusions Resistance to BRAF inhibition is mediated by reactivation of the MAPK pathway in a CRAF-dependent manner. Pan-RAF inhibitors synergize with MEK inhibitors to suppress proliferation and induce apoptosis in both BRAF mutant, PLX4720-resistant cell lines and also in KRAS mutant cell lines. Pan-RAF inhibitors are progressing into clinical trials and our data support their use in combination with MEK inhibitors. Citation Format: Steven R. Whittaker, Glenn S. Cowley, Steve Wagner, Flora Luo, David E. Root, Levi A. Garraway. Combined pan-RAF and MEK inhibition overcomes multiple resistance mechanisms to selective RAF inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C72.