Abstract 7020: Chemical probe discovery for PAX3::FOXO1 via small-molecule microarrays

Abstract PAX3::FOXO1 (P3F) oncoprotein is a driver of poor patient outcome, contributing to tumorigenesis by dysregulating protein complexes that result in altered chromatin structure and gene expression. We conducted Small-Molecule Microarray (SMM) screening of approximately 10, 000 compounds using engineered HEK293 cell lysates, identified KI-P3F-032, a compound that binds silently to the fusion protein, forming the basis for PROTACs development. With this initial results, there is a need to revalidate this preliminary screening result with biophysical data, we validated binding with SPR and developed biologically relevant high-throughput secondary assays to evaluate PROTACs efficacy. We have now developed a PROTAC with strong selective cytotoxicity against P3F positive RMS cell lines. At the same time, we also explored the possibility of indirect targeting of P3F via inhibiting and degrading other transcription regulators such as CDK9. We also developed new SMM assays to identify novel P3F binders using a library of approximately 65, 000 small molecules under native P3F positive cell lysates, identifying 90 potential binders. We then developed functional recombinant P3F protein production and purification in our lab to enable pure protein based assays. We hope our research strategy can further advance the current progress on targeting the conventionally undruggable P3F oncogenic fusion and bring insight to targeting disordered oncogenic fusions in other pediatric cancers. Citation Format: Yichen Xiang, Marc Ramos Sala, Bocheng Wu, Diya Rana, Keisuke Motoyama, Becky Leifer, Mohammed Toure, Shelby Doyle, Angele N. Koehler. Chemical probe discovery for PAX3::FOXO1 via small-molecule microarrays [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 7020.