Abstract Introduction: Ewing sarcoma (ES) is an aggressive pediatric bone and soft tissue cancer that is absolutely dependent on the EWS::FLI1 fusion transcription factor. Despite this dependency, ES tumors demonstrate substantial variability in EWS::FLI1 transcriptional activity both across tumors and within individual tumors. Here, we investigate the inter- and intra-tumoral heterogeneity of EWS::FLI1 and its functional importance. Methods: We established a highly optimized siRNA knockdown protocol for EWS::FLI1 in 6 preclinical ES cell line models to achieve equal levels of suppression and evaluate differences in induced and repressed transcriptional targets and DNA binding events of EWS::FLI1. In order to investigate underlying mechanisms driving resistance, we employed state-of-the-art techniques including Cleavage Under Target (CUT Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 635.
Hinshaw et al. (2026) studied this question.