Abstract 1080: Using genome-wide CRISPR screening to develop novel combination therapies for high-risk Ewing’s sarcoma

Abstract Ewing’s sarcoma (EWS) is an aggressive bone and soft-tissue cancer, characterized by a hallmark fusion oncoprotein comprising the amino terminus of a FET gene and the carboxy terminus of an ETS gene, most commonly (85%), the EWSR1-FLI1 t (11, 22) fusion. TK-216 is a first-in-class EWSR1-FLI1 inhibitor currently in phase II clinical trials for EWS. Although TK-216 was designed to target the fusion protein interaction directly, it has demonstrated limited efficacy as a monotherapy. Thus, more effective TK-216 combination therapies are needed. We have used genome wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Knockout screening in EWS cells treated with TK-216 to identify novel synthetic lethality. We have identified 97 genes whose deletion potentially enhances the action of TK-216 in EWS cells. We have also identified several druggable targets, such as USP9X and the oxidative phosphorylation (OXPHOS) pathway as synthetic lethal interactions for TK-216. USP9X is known to deubiquitinate and stabilize the EWS-FLI1 fusion protein. USP9X and EWS-FLI1 protein expression levels were found to be positively correlated in EWS clinical specimens. USP9X siRNA-mediated gene silencing reduced EWS cell viability. Most importantly, USP9X siRNA-mediated gene silencing sensitized EWS cells to TK-216 treatment. In addition, inhibition of USP9X with a small-molecule inhibitor WP1130, led to significant cell death in EWS alone and in combination with TK-216 with wide therapeutic indices. The molecular mechanism of synergy and in vivo efficacy of the novel TK-216 combination will be determined in our study for future clinical translation. Citation Format: Daenikka Ravindrarajah, Sukriti Krishan, Kayleen Simpson, Twishi Gulati, Belamy B. Cheung, Glenn M. Marshall. Using genome-wide CRISPR screening to develop novel combination therapies for high-risk Ewing’s sarcoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1080.