Abstract 5727: Identification of synthetic lethal vulnerabilities in cancers with loss of function mutations in NOTCH

Loss of Function (LOF) mutations in the NOTCH receptors are found in several different cancers most notably in small cell lung cancer (SCLC) and squamous cell carcinomas (SCC). We previously developed genetically engineered mouse models (GEMM) of SCLC using CRISPR/Cas9 engineered to be NOTCH1-Mutant, NOTCH2-Mutant, and NOTCH-WT. Synthetic lethality provides a paradigm for targeting cancers with LOF mutations in tumor suppressor genes. In applying this paradigm, one looks for specific vulnerabilities that are created upon loss of the gene of interest. Using 6 cell lines developed from these CRISPR-based SCLC GEMMs which are isogenic to NOTCH, we performed CRISPR/Cas9 LOF negative selection screens (using an sgRNA library enriched in druggable enzymes) to identify synthetic lethal interactors with LOF NOTCH mutations. Our CRISPR/Cas9 screen identified TRIM28 (Tripartite Motif Containing 28, or KAP1) as a highly significant synthetic lethal interactor with NOTCH1 or NOTCH2. We validated the synthetic lethal interaction between NOTCH1/2 and TRIM28 using both human and mouse NOTCH-isogenic cell lines. Interestingly, RNA-sequencing of NOTCH2-Mutant or NOTCH-WT SCLC cell lines after TRIM28 CRISPR inactivation showed robust increases in endogenous retroviral (ERV) expression and the MDA5/RIG-I/MAVS RNA-sensing machinery leading to hyperactivation of the TBK1/IRF3/7 innate immune signaling pathway and the pro-inflammatory cytokine CXCL10 selectively in cells with NOTCH inactivated. Notably, inactivation of TBK1 activity or JAK-STAT activity using specific small molecule inhibitors of TBK1 or JAK completely reversed the synthetic lethality between NOTCH and TRIM28 suggesting that the synthetic lethality phenotype is a consequence of hyperactivation of innate immune signaling, which is known to be cytotoxic. Together our findings uncover a novel vulnerability in SCLCs with LOF NOTCH mutations and perhaps other cancers with LOF NOTCH mutations and suggest a strategy that could also potentiate anti-tumor immunity by increasing innate immune signaling in tumor cells. Citation Format: Deli Hong, Matthew A. Booker, Sidrah Anjum, Yixiang Li, Tran C. Thai, Michelle Y. Wang, David A. Barbie, Michael Y. Tolstorukov, Jun Qi, Matthew G. Oser. Identification of synthetic lethal vulnerabilities in cancers with loss of function mutations in NOTCH. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5727.