Abstract B108: A genome-scale shRNA synthetic lethal screen identifies enhancers of sensitivity to RAF inhibition.

Genome-scale RNA interference screens have identified a dependency on BRAF for the proliferation of BRAF-mutant colorectal cancer (CRC) cell lines. Despite this, small molecule RAF inhibitors, such as vemurafenib, have been unsuccessful in clinical trials of patients with CRC, with a response rate of only 5%. CRC cell lines demonstrate intrinsic resistance to RAF inhibitors, in some cases through EGFR or MET activity. We designed a genome-scale RNA interference screen to identify loss of function events that could synergize with pharmacologic RAF inhibition. We transduced a pooled lentiviral library encoding 90,000 shRNAs targeting over 16,500 genes into the RKO CRC cell line, which displays robust resistance to PLX470. The shRNA-infected population was divided into two experimental arms, one treated with DMSO, the other treated with PLX4720. Following 16 population doublings, the abundance of each hairpin was assessed by PCR amplification of barcoded hairpin DNA, followed by massively parallel paired-end sequencing. The log-fold change in shRNA abundance between the PLX4720- and DMSO-treated controls was determined and RNAi gene enrichment (RIGER) used to rank individual shRNAs and nominate candidate genes that were required for survival of RKO cells exposed to PLX4720. Top-ranking genes that displayed synthetic lethality with RAF inhibition were validated in an arrayed secondary screen. shRNAs targeting MET scored highly, consistent with recent reports implicating HGF-MET signaling in resistance to BRAF inhibition, validating our approach. Furthermore, we describe the characterization of additional genes that sensitize cells to inhibition of BRAF. Ultimately, these studies aim to nominate pharmacologically targetable events that mediate RAF-inhibitor resistance in colorectal cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B108. Citation Format: Steven R. Whittaker, Flora Luo, Jessica Hsiao, Glenn S. Cowley, David E. Root, Levi A. Garraway. A genome-scale shRNA synthetic lethal screen identifies enhancers of sensitivity to RAF inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B108.