Abstract B013: EWS::FLI1 activity at GGAA repeats is essential for maintenance of Ewing-like splicing

Abstract Background: Ewing sarcoma is a pediatric bone and soft tissue cancer thought to arise from mesenchymal stem cells (MSCs). It is characterized by fusion of the RNA-binding protein EWS to an ETS family transcription factor, most often FLI1. EWS::FLI1 induces changes in gene expression that have been well-studied and have been linked to its ability to bind to and alter chromatin state at GGAA repeat sequences within the genome. In addition to these effects, EWS::FLI1 alters mRNA splicing; however, the mechanism of this activity remains unclear. Methods: We curated a sarcoma-focused transcriptome from published long-read RNA sequencing of Ewing sarcoma, osteosarcoma, and synovial sarcoma patient tumors and matched adjacent normal tissue (215M reads). We supplemented this with published long-read RNA sequencing data from relevant normal cell types, osteosarcoma cell lines, and Ewing sarcoma lines with or without EWS::FLI1 knockdown (105M reads). We used this transcriptome as a reference to evaluate expression and splicing from short-read RNA-seq of Ewing sarcoma cell lines, MSCs, and MSC-based models of Ewing sarcoma and established Ewing-associated expression and splicing signatures. Results: We generated scores based on these signatures in published knockdown-rescue experiments in which endogenous EWS::FLI1 was silenced by shRNA and replaced with exogenous shRNA-resistant mutants. Expression and splicing scores were highly correlated within these datasets (r2 = 0.89), suggesting that similar regions of the fusion protein might be required for both its functions. In contrast, the scores had limited correlation within an internal dataset of RNA-binding protein perturbations (r2 = 0.02). We next examined the effects of pharmacological agents thought to alter EWS::FLI1 activity. Interestingly, we found that lurbinectedin which disrupts EWS::FLI1’s DNA-binding could partially revert both expression and splicing scores. In contrast, YK-4-279 which disrupts the fusion’s protein-protein interactions and MS0621 which reverts a Ewing-associated chromatin signature had limited effects on these scores. To further interrogate the potential role of EWS::FLI1 DNA-binding in splicing regulation, we silenced EWS::FLI1 in TC-32 cells and replaced it with either the wildtype fusion or a DNA-binding null mutant. Unlike the wildtype construct, the DNA-binding null mutant could not rescue expression or splicing. Finally, we examined the effects of the previously published KRAB-ZFA7 repressor which silences chromatin at GGAA repeats while sparing single GGAA sites and found that it could revert both scores. Discussion: Together, these data establish a potential connection between EWS::FLI1 activity at GGAA repeats and its effects on mRNA splicing. Additionally, we find that lurbinectedin, an FDA-approved agent currently in a phase 1/2 trial for Ewing sarcoma, suppresses both the splicing and expression effects of EWS::FLI1. Emerging compounds designed to target EWS::FLI1 DNA-binding directly will likely have similar effects. Citation Format: David V. Allegakoen, Jeffrey A. Toretsky. EWS::FLI1 activity at GGAA repeats is essential for maintenance of Ewing-like splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B013.