Abstract 3109: Protein stability screen to identify compounds destabilizing EWS-FLI1 in Ewing sarcoma

Abstract

Abstract Ewing sarcoma is an aggressive pediatric bone and soft tissue tumor driven by the expression of a fusion oncoprotein named EWS-FLI1, which acts as an oncogenic transcription factor. Tumor cells are strictly dependent on continuous expression of the fusion protein, since downregulation of EWS-FLI1 inhibits tumor growth. Therefore, interference with the fusion oncoprotein turnover is critical for the modulation of tumor cell proliferation and survival. We demonstrated previously that EWS-FLI1 is predominantly a proteasomal substrate with high turnover mediated by poly-ubiquitination at one specific lysine. This study provided novel insights into the crucial importance of targeting EWS-FLI1 stability as novel strategy for the treatment of Ewing sarcoma. Hence, we aimed at identifying compounds that destabilize EWS-FLI1 with subsequent reduction of tumor cell growth by performing a screen of 2’486 FDA-Approved drugs and 204 novel targeted compounds in a Ewing sarcoma reporter cell line. To this, we adopted a Global Protein Stability approach as novel read-out (Global protein stability profiling in mammalian cells, Science, 2008), which relies on a reporter construct expressing two fluorescent dyes from one mRNA to monitor changes in stability of EWS-FLI1 by high-throughput flow cytometry. This drug screen identified two main enriched classes of inhibitors that significantly destabilize EWS-FLI1. Validation of these results and characterization of their mechanism of action is currently under way. We conclude that the study of EWS-FLI1 turnover represents a novel approach to identify new effective drugs that can be used as monotherapy or in combination with other drugs as novel treatment opportunities in Ewing sarcoma. Citation Format: Gloria Pedot, Felix K. Niggli, Beat W. Schaefer. Protein stability screen to identify compounds destabilizing EWS-FLI1 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3109.