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

Area of Science:

• Oncology
• Molecular Biology
• Drug Discovery

Background:

• Fusion-positive rhabdomyosarcoma (FP-RMS) is driven by specific chromosomal translocations, notably PAX3::FOXO1.
• The PAX3::FOXO1 fusion protein is crucial for FP-RMS initiation and maintenance, representing a key therapeutic target.
• FP-RMS generally has poorer survival outcomes compared to fusion-negative cases.

Purpose of the Study:

• To develop and validate Proteolysis Targeting Chimeras (PROTACs) capable of degrading the oncogenic PAX3::FOXO1 fusion protein.
• To assess the efficacy of PAX3::FOXO1-targeting PROTACs in FP-RMS cell lines.
• To explore the potential of PROTACs as a therapeutic strategy for FP-RMS.

Main Methods:

• Creation of PROTACs by linking PAX3::FOXO1-binding molecules with E3 ligase recruiters (CRBN or SKP1).
• Evaluation of PROTAC-mediated PAX3::FOXO1 degradation in FP-RMS cell lines.
• Assessment of downstream effects, including gene expression changes, myogenic differentiation, and synergy with vincristine.

Main Results:

• PROTACs achieved up to 70% degradation of endogenous PAX3::FOXO1 in FP-RMS cells.
• Degradation was concentration-, time-, and proteasome-dependent.
• PROTAC treatment induced myogenic differentiation, altered gene expression, and synergized with vincristine to inhibit anchorage-independent growth (>80%).

Conclusions:

• Demonstrated proof of principle for PROTACs targeting PAX3::FOXO1 in FP-RMS.
• Developed PROTACs serve as valuable tools for studying PAX3::FOXO1 biology.
• These PROTACs provide a molecular scaffold for developing clinical therapeutics for FP-RMS patients.