Abstract A12: CRISPR-mediated modeling and functional validation of candidate tumor-suppressor genes in small cell lung cancer

Abstract

Abstract Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified a large number of recurrently mutated genes in human SCLC tumors. However, apart from a few notable examples, the functional roles of most of these genes remain to be validated. The CRISPR-Cas9 system has been utilized in genetically engineered mouse models of cancer to generate somatic genetic alterations in tumor cells in vivo. This has greatly accelerated efforts to study the roles of candidate genes of interest in various cancers. In this study, we adapted the CRISPR-Cas9 system to murine models of SCLC (mSCLC) to enable rapid modeling and functional validation of candidate tumor-suppressor genes in SCLC. We generated a Cre-activated allele of Cas9 and crossed it into both the Trp53flox/flox; Rb1flox/flox and the Trp53flox/flox; Rb1flox/flox; p130flox/flox models of mSCLC. By infecting these animals with adenoviral vectors coexpressing Cre recombinase and a target sgRNA, we can initiate SCLC tumors harboring loss-of-function mutations in candidate genes of interest. To validate this system, we infected animals with adenoviral vectors expressing sgRNAs targeting either p107 (Rbl1) or p130 (Rbl2), two genes that are recurrently mutated in a subset of human SCLC tumors. SCLC tumors that developed in these animals harbored loss-of-function mutations at the respective sgRNA target sites in the genome. Loss of p107 or p130 expression in these tumors was confirmed by immunohistochemical staining. As expected, CRISPR-mediated loss of p130 in Trp53flox/flox; Rb1flox/flox animals resulted in acceleration of tumor progression compared with animals infected with a control sgRNA, consistent with the phenotype previously observed in Trp53flox/flox; Rb1flox/flox; p130flox/flox animals. Loss of p107 in Trp53flox/flox; Rb1flox/flox animals also resulted in acceleration of tumor progression, as seen by an increase in tumor burden and a decrease in overall survival compared with control animals. Such an acceleration was not observed with loss of p107 in Trp53flox/flox; Rb1flox/flox; p130flox/flox animals, suggesting potential functional redundancy between p107 and p130 in SCLC. In summary, we have demonstrated the feasibility of using the CRISPR-Cas9 system to rapidly model loss-of-function mutations in candidate tumor-suppressor genes in mSCLC, and have used this approach to validate the role of p107 as a tumor suppressor in SCLC. Such an approach will greatly accelerate efforts to investigate the molecular mechanisms of tumor progression in SCLC. Citation Format: Sheng Rong Ng, William M. Rideout, III, Bethany L. Wagner, Tyler Jacks. CRISPR-mediated modeling and functional validation of candidate tumor-suppressor genes in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A12.