PAX3::FOXO1-Targeting PROTAC Induces Myogenic Differentiation of Fusion-Positive Rhabdomyosarcoma Cells.

Background/Objectives: Fusion-positive rhabdomyosarcoma (FP-RMS) is characterized by the presence of tumor-specific chromosomal translocation products, most commonly PAX3::FOXO1, and typically results in lower survival rates compared to fusion-negative RMS cases. PAX3::FOXO1 plays a critical role in FP-RMS oncogenesis in both tumor initiation and maintenance, making it an excellent target for therapeutic intervention in FP-RMS. Methods: We created Proteolysis Targeting Chimeras (PROTACs) by combining PAX3::FOXO1-binding small molecules with E3 ligase recruiters for cereblon (CRBN) or S-Phase Kinase Associated Protein 1 (SKP1). Results: The PROTACs achieved up to 70% degradation of the endogenous PAX3::FOXO1 protein in FP-RMS cell lines in a concentration-, time-, and proteasome-dependent manner. Moreover, the PROTAC-mediated targeted degradation of PAX3::FOXO1 in FP-RMS cells deregulated the endogenous PAX3::FOXO1 gene expression signature and induced myogenic differentiation. Importantly, treatment of FP-RMS cells with PAX3::FOXO1-PROTACs synergized with vincristine treatment and impaired >80% of anchorage-independent growth in soft agar. Conclusions: Taken together, we demonstrate the proof of principle of PROTACs targeting the oncogenic fusion protein PAX3::FOXO1 in FP-RMS cells. The PROTACs created in this study will not only be useful tools in studying PAX3::FOXO1 biology in laboratory models but could also serve as molecular scaffolds for designing clinical-grade molecules to assess the therapeutic potential of PAX3::FOXO1-targeting PROTACs in FP-RMS patients.