Dual inhibition of KDM4B and KDM5A disassembles the PAX3-FOXO1 transcriptional program in fusion-positive rhabdomyosarcoma

Fusion-positive rhabdomyosarcoma (FP-RMS) is driven by the oncogenic transcription factor PAX3-FOXO1 and is associated with poor clinical outcome. The histone demethylase KDM4B has been implicated in sustaining PAX3-FOXO1-dependent transcriptional programs, but the epigenetic regulatory mechanisms supporting this network remain incompletely characterized. We employed a deep learning-based drug discovery strategy and identified a novel small-molecule, Compound 01. Functional and mechanistic analyses were then performed to delineate cooperative interactions among KDM4B, KDM5A, and PAX3-FOXO1, and to evaluate the impact of pharmacological perturbation on the stability of this transcriptional network in FP-RMS models. In this study, we identified Compound 01 with dual activity against KDM4B and KDM5A that triggers proteasome-dependent loss of both proteins. Mechanistically, we uncover a reciprocal stabilization loop in which KDM4B, KDM5A, and PAX3–FOXO1 reinforce each other to sustain the PAX3–FOXO1 transcriptional state. Pharmacological disruption of this axis by Compound 01 collapses the PAX3–FOXO1-driven oncogenic transcriptional program, suppresses key downstream targets, and markedly impairs FP-RMS cell proliferation and metastatic potential. These findings identify KDM5A as a critical epigenetic partner of the PAX3–FOXO1 network and establish dual KDM4B/KDM5A targeting as a strategy to destabilize PAX3–FOXO1 transcriptional circuitry in FP-RMS.