Abstract PR12: Genome-wide in-vivo tumor xenograft CRISPR knockout screening for identifying KRAS mutant synthetic lethal interactions (2017)

Abstract The KRAS oncogene is frequently mutated in many of the most lethal human cancers and has been resistant to targeted therapies, leading to efforts to identify synthetic lethal genetic interactions in large-scale screens. These screens have been carried out largely in in-vitro cell culture systems using RNA interference (RNAi). Functional genomic screening using the bacterial type II clustered regularly interspaced short palindrome repeats and their associated proteins (CRISPR-Cas9) system has proven to be more powerful than RNAi for systematic genomic perturbation due to its simplicity, decreased off-target effects, and the ability to create knockout rather than knockdown phenotypes. We have conducted a genome-wide CRISPR-Cas9 loss-of-function screen with the human GeCKO v2 library directly in-vivo utilizing a paired isogenic human colorectal cell line (HCT116) with and without a KRAS oncogenic mutation (G13D) to identify genes whose knockout results in synthetic lethal interactions in tumor xenografts with mutated KRAS. Compared to cell culture models, tumor xenografts better recapitulate obstacles tumor cells must overcome for continued proliferation including limited accessibility to nutrients and oxygen. Many recent studies have also highlighted the effect of KRAS oncogenic mutations on the rewiring of metabolic pathways to fuel increased tumor growth. Tumor xenografts offer a better screening platform in identifying factors that might exploit these metabolic pathways. Pathway analysis of our genome-wide CRISPR screen indeed revealed candidate synthetic lethal genes involved in metabolic and nucleic acid synthesis pathways in KRAS mutant tumor xenografts. We individually validated some of the candidate genes in these metabolic pathways (including the TCA cycle, pentose phosphate pathway, and fructose metabolism) with individual CRISPR-Cas9 knockouts and confirmed that knockout of these genes resulted in decreased tumor xenograft growth in KRAS mutant tumors. One of the KRAS synthetic lethal genes that we identified was the gene NADK, which encodes NAD+ kinase that converts NAD+ into NADP+. Low-frequency activating mutations in NADK were also recently discovered by another group to harbor oncogenic potential in pancreatic adenocarcinoma. Given the high frequency of KRAS mutations in pancreatic adenocarcinoma, NADK may represent a therapeutic target in KRAS mutant tumors in general. While more sensitive than RNAi screens, there was still significant noise in our CRISPR-Cas9 genome-wide screen as expected in an in-vivo dropout screen. To overcome this noise, we generated a smaller pooled lentiviral CRISPR library (targeting ~250 genes) based on candidate hits in the genome-wide screen and again directly screened this smaller but deeper pooled library in tumor xenografts with higher coverage and more replicates. This strategy allowed for better discrimination of novel KRAS dependent synthetic lethal genes. Citation Format: Edwin H. Yau, Tariq Rana. Genome-wide in-vivo tumor xenograft CRISPR knockout screening for identifying KRAS mutant synthetic lethal interactions [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR12.