Mentor Name: Jeffrey Toretsky

Ewing Sarcoma is an aggressive bone and soft tissue cancer driven by the EWS::FLI1 fusion protein, an irregular transcription factor that alters gene expression in cells, effectively promoting tumor growth and metastasis. Given the poor outcomes for patients with relapsed or metastatic disease, EWS::FLI1 serves as a pertinent target for the development of novel, targeted interventions. Due to the fact that EWS::FLI1 is an intrinsically disordered protein lacking stable binding pockets, directly inhibiting this oncogenic protein has proved to be challenging. However, recent discoveries of crucial partner proteins of EWS::FLI1 have become effective targets of small molecule inhibitors that interrupt these protein-protein interactions. In particular, the inhibitor TK216 has been shown to disrupt the interactions between EWS::FLI1 and RNA helicase A (RHA), inducing apoptosis in treated cells. While TK216 demonstrates strong therapeutic potential in targeting EWS::FLI1 protein interactions with RHA, additional compounds that target different protein partners are needed in order to expand treatment options and improve efficacy. As such, this project aims to evaluate four candidate small molecule inhibitors previously identified using AlphaFold3 and surface plasmon resonance assays to bind to EWS::FLI1. This study will utilize three established Ewing Sarcoma cell lines, A4573, SKES, and TC32 cells, to evaluate whether drug responses are consistent across different EWS::FLI1 fusion types. This project will investigate drug-induced changes in cell viability and tumorigenic behavior as well as immunoprecipitation of known EWS::FLI1 protein partners such as RHA, p68, and hnRNP K. Ultimately, these assays will determine whether the identified small molecule inhibitors disrupt EWS::FLI1-mediated oncogenic activity and the possible mechanisms by which it does so. Overall, the end goal is to contribute to the development of a more effective and targeted therapy for Ewing Sarcoma patients.