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

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. 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). PROTACs achieved degradation of endogenous PAX3::FOXO1 protein in FP-RMS cell lines in a concentration-, time-, and proteasome-dependent manner. Moreover, bulk RNA-seq revealed that PAX3::FOXO1-targeting PROTAC treatment impaired endogenous PAX3::FOXO1 target gene expression and induced myogenic differentiation. Importantly, treatment of FP-RMS cells with PAX3::FOXO1-PROTACs synergized with vincristine treatment and demonstrated an impairment in anchorage-independent growth in soft agar. Taken together, we demonstrate the proof of principle of PROTACs targeting the oncogenic fusion protein PAX3::FOXO1 in FP-RMS cells. PROTACs created in this study will not only be useful tools in studying PAX3::FOXO1 biology in laboratory models but could also serve as scaffolds for designing clinical-grade molecules to assess the therapeutic potential of PAX3::FOXO1-targeting PROTACs in FP-RMS patients.