Abstract 623: Targeting PAX3::FOXO1 condensates in fusion-positive alveolar rhabdomyosarcoma via disordered-region mimetic peptides.

Abstract Background: PAX3::FOXO1 (P3F1) is a fusion oncoprotein that drives fusion-positive alveolar rhabdomyosarcoma (aRMS), a highly lethal pediatric cancer with 30% 5-year survival in high-risk patients. Targeted therapy development has been limited by P3F1’s largely disordered structure and poorly understood interaction mechanisms. Recent work has shown that short peptides targeting intrinsically disordered regions (IDRs) can disrupt transcriptional hubs and impair tumor growth, but this strategy requires identifying key interaction partners that often regulate other signaling pathways. We previously discovered that P3F1 demonstrates a neomorphic hub formation propensity that corresponds with its transcriptional activity, suggesting that disrupting these hubs can hinder said activity. Purpose: To design short peptides modelled from the P3F1 IDR itself that disrupt P3F1 hubs and suppress P3F1-mediated transcription and aRMS cell proliferation. Experimental Procedures and Results: Using the LacO array assay, an established in-cellulo method that quantifies IDR interaction strength, we discovered that truncating the P3F1 392-510 region significantly decreased IDR interaction strength. Further, the same truncation significantly altered its interaction strength with p300, a key transcriptional coactivator known to promote P3F1’s function. Finally, dual luciferase assay results showed that a 392-510 deletion significantly decreased P3F1’s transactivation ability. Taken together, this suggested that P3F1 392-510 is a key interaction hotspot that influences its transcriptional capability. Sequence analysis revealed that P3F1 392-510 is enriched in cationic and aromatic residues, consistent with cation-π and π-π interactions common in IDRs. FINCHES interaction mapping showed that mutating either all cationic or all aromatic residues in this region reduces P3F1 homotypic interactions, with aromatic-to-alanine mutations producing the stronger effect. Ongoing experiments utilizing the LacO array assay are probing these interaction modes to guide the design of optimized P3F1 392-510 peptide mimics. Conclusions: P3F1 392-510 is critical for P3F1’s IDR-mediated interactions and transcriptional activity. Its cationic and aromatic composition suggests cation-π and/or π-π interactions as key interaction modes. Future work will experimentally dissect these interaction modes and develop peptide inhibitors informed by this mechanistic insight. Citation Format: Michael Di Martino, Yanghao Zhong, Shasha Chong. Targeting PAX3::FOXO1 condensates in fusion-positive alveolar rhabdomyosarcoma via disordered-region mimetic peptides abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 623.