Abstract B27: Probing synthetic lethal interactions with RAF inhibition in BRAF-mutant colorectal cancer

• Published in: Molecular cancer therapeutics Vol. 12; no. 5_Supplement; p. B27
• Main Authors: Whittaker, Steven R., Luo, Flora, Hsiao, Jessica, Cowley, Glenn S., Root, David E., Garraway, Levi A.
• Format: Journal Article
• Language: English
• Published: 01.05.2013
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.PMS-B27

Abstract Large-scale RNA interference screens have indicated that BRAF-mutant colorectal cancer (CRC) cell lines are dependent upon BRAF expression for proliferation. However, small molecule RAF inhibitors, such as vemurafenib, have so far failed to exhibit robust activity in clinical trials of patients with CRC, with a response rate of only 5%. Commensurate with this, CRC cell lines demonstrate intrinsic resistance to RAF inhibitors. Therefore, we designed an 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 RAF-inhibitor-resistant RKO CRC cells. 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. We validated the 40 top-ranking genes that displayed synthetic lethality with RAF inhibition 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, suggesting novel therapeutic opportunities. By integrating these findings with other genomic and functional studies we aim to identify clinically actionable events that cause RAF-inhibitor resistance in colorectal cancers. Citation Format: Steven R. Whittaker, Flora Luo, Jessica Hsiao, Glenn S. Cowley, David E. Root, Levi A. Garraway. Probing synthetic lethal interactions with RAF inhibition in BRAF-mutant colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B27.