Multi-omics analyses identify EZH2 as a central driver in rhabdomyosarcoma radioresistance and highlight Tazemetostat as an effective radiosensitizer in vitro and in vivo

: Rhabdomyosarcoma (RMS), a pediatric soft tissue sarcoma, comprises two major subtypes: fusion-positive (FP), driven by PAX3/7-FOXO1 fusions, and fusion-negative (FN), often harboring RAS pathway mutations. High-risk RMS exhibits intrinsic resistance to radiotherapy (RT), posing a significant clinical hurdle. Emerging evidence implicates EZH2, the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), in promoting RT resistance through gene silencing via H3K27me3. To dissect the molecular basis of RMS radioresistance, we employed an integrative multi-omics approach encompassing phosphoproteomics and transcriptomics. Radioresistant RMS cell models (RMSCRR) displayed enhanced cancer stem cell features, evasion of RT-induced G2/M arrest, and reduced apoptosis compared to their parental counterparts (RMSPR). Phosphoproteomic profiling revealed activation of prosurvival and proliferative pathways across both FN and FP subtypes. Transcriptomic analysis identified a robust downregulation of EZH2 target genes, with distinct gene sets modulated in FN-RMSCRR versus FP-RMSCRR cells, highlighting subtype-specific epigenetic rewiring. These multi-omics findings pointed to hyperactive PRC2/EZH2 signaling as a driver of radioresistance. Therapeutically, combining the EZH2 inhibitor Tazemetostat (TZM) with RT significantly impaired clonogenic survival, enhanced G2/M arrest, and promoted apoptosis in both RMSPR and RMSCRR cells. In vivo, RT and TZM co-treatment fully suppressed FN-RMSPR tumor growth and delayed FP-RMSPR progression. Notably, TZM monotherapy inhibited tumor growth in both FN- and FP-RMSCRR xenografts, uncovering a therapy-induced vulnerability. Our integrative multi-omics analysis reveals EZH2-dependent molecular programs underpinning radioresistance and supports EZH2 targeting as a rational radiosensitizing and therapeutic strategy in RMS, including recurrent and RT-refractory disease.