The fusion transcription factor PAX3-FOXO1 (P3F) is the major driver of alveolar rhabdomyosarcoma. Since these tumors are characterized by a mostly quiet mutational landscape and a paucity of druggable oncogenes, the fusion protein itself remains the most important drug target. Transcription factors are challenging proteins for drug development since they lack enzymatic activities and are, apart from DNA binding domains, largely intrinsically disordered. Identification of defined and possibly druggable structures in such proteins is therefore of great clinical interest. Towards this aim, we performed a CRISPR/Cas9-based domain screen in P3F-positive rhabdomyosarcoma cells and identified a small, structured and highly important domain within the transactivation domain of P3F. Further, we demonstrate that cysteine C793 located in this region is indispensable for target gene activation by P3F. Its mutation significantly reduced cell proliferation and induced differentiation of RMS cells. Mechanistically, we identified p300/CBP proteins as important co-factors and interactors of C793 that co-regulate a majority of P3F target genes. Their inhibition/degradation by small molecules efficiently reduces rhabdomyosarcoma cell survival. These data suggest that mainly one single amino acid drives oncogenicity of P3F and identify a potentially targetable structure within the fusion protein.

Citation Format: Beat W. Schäfer, Katharina Benischke, Jakob Wurth, Dominik Laubscher, Quy A. Ngo, Sara Danielli, Marco Wachtel. A single cysteine in PAX3-FOXO1 is relevant for transactivation and survival of rhabdomyosarcoma cells [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR007.