Abstract 1048: Synergistic drug combination of LSD1 and ABL2 inhibitors for Ewing's sarcoma identified in an arrayed CRISPR screen approach

Abstract

Abstract Background: Ewing's Sarcoma (ES) is a rare and aggressive pediatric bone tumor that is driven by a chromosomal translocation and fusion between EWS and FLI1 genes resulting in a chimeric protein. Nearly 85% of ES cases express the EWS/FLI protein which functions as a transcription factor and drives oncogenesis. EWS/FLI interacts with lysine-specific demethylase 1 (LSD1) that is highly expressed in pediatric sarcomas, to repress critical tumor suppressors. We have developed a potent reversible LSD1 inhibitor, SP-2577 (Seclidemstat), that shows the ability to impair tissue culture cell viability in multiple ES cell lines and is currently in clinical trials. A more favorable therapeutic response to SP-2577 may be obtained by combining drugs that target multiple pathways and/or inhibit resistance mechanisms. Experimental Design: In this study, we applied an arrayed CRISPR screening (Synthego) to identify survival genes that can be candidates for molecularly targeted drugs. Gene candidates were validated using western blot, qPCR, soft agar assay. Ewing’s Sarcoma A673 ABL2i knockdown and scramble cell lines were generated, implanted into nude mice, and treated with SP-2577. Lastly, combinational drug treatments using FDA-approved inhibitors with SP-2577 were tested in 2D and 3D cultures. Results: CRISPR druggable library screen identified that downregulation of several genes resulted in increased SP-2577 cytotoxicity in multiple ES cell lines. Importantly, ABL2 knockdown was found to have a significant synergy in combination with SP-2577 treatment, both in vitro and in vivo. siRNA knockdown of ABL2 in combination with SP-2577 treatment revealed an increase in JNK signaling and Cleaved Caspases-3 activation with decreased Src phosphorylation. This data suggests that downregulation of ABL2 in combination with SP-2577 treatment promotes activation of apoptosis pathways in ES. In addition, combination treatment of SP-2577 with drugs targeting ABL2 pathway showed high synergy in vitro. Conclusion: This study highlights the potential for a combination treatment of SP-2577 with ABL2 inhibitors in ES patients. Further, our observations support the capability of arrayed CRISPR screening to identify new therapeutic strategies in ES. Citation Format: Samuel Sampson, Trason Thode, Kevin Drenner, Ryan Rodriguez Del Villar, Annika Ozols, Mohan Kaadige, Alexis Weston, Serina Ng, Tithi Ghosh Halder, Shelby Rheinschmidt, David Anderson, Kevin Holden, Raffaella Soldi, Sunil Sharma. Synergistic drug combination of LSD1 and ABL2 inhibitors for Ewing's sarcoma identified in an arrayed CRISPR screen approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1048.