Abstract 5488: Identifying novel Ewing sarcoma therapeutics using a high-throughput chromatin accessibility assay

Abstract Chromatin dysregulation has been implicated in the development of a wide range of diseases, including cancer, as well as therapeutic resistance. Genetic alterations in chromatin regulatory factors and aberrant activity of transcriptional regulators alter chromatin states and drive tumor growth in cancer. One example of a cancer that is driven by alterations in chromatin landscape is Ewing sarcoma, a highly aggressive pediatric cancer of bones and soft tissues. Ewing sarcoma is driven by an oncoprotein generated by the translocation of the EWSR1 and FLI1 genes, resulting in the fusion oncoprotein (EWSR1::FLI1), which activates noncanonical enhancers and alters the expression of critical genes associated with the disease. EWSR1::FL1 is challenging, if not impossible, to target directly with small molecules, and viable therapeutic targets for Ewing sarcoma have been slow to emerge. To broaden the search for druggable targets we developed a target agnostic screening approach that exploits oncoprotein-specific chromatin accessibility states as a relevant and direct functional readout for identifying novel therapeutics. We developed and optimized a 384-well, high throughput (HT) version of the Assay for Transpose-Accessible Chromatin assay (HT-ATAC-seq) that was applied to 500 EWSR1::FLI1-regulated chromatin regions. Using Ewing sarcoma-derived A673 cells, we conducted a pilot screen of 12,000 small-molecule compounds comprising approved drugs, investigational agents, and bioactive compounds. The screen identified 285 compounds that partially reversed Ewing sarcoma chromatin abnormalities. The set of active compounds included Panobinostat, which is currently in preclinical development for Ewing sarcoma. This pilot screen supports the HT-ATAC-seq assay as a robust approach to identify novel compounds that reverse the chromatin alterations associated with Ewing sarcoma. This approach and assay can be adapted to investigate other cancers or pathologies associated with chromatin state alterations. Citation Format: Darian Williams, Andrew McFadden, Austin Hepperla, Min Shen, Tino Sanchez, Gordon Stott, Mark Henderson, Ian Davis, Samantha Pattenden. Identifying novel Ewing sarcoma therapeutics using a high-throughput chromatin accessibility assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5488.