(P 400) LONGITUDINAL EPIGENETIC AND TRANSCRIPTOMIC REMODELING ACROSS PRIMARY, IRRADIATED, AND LOCALLY RELAPSED SOFT TISSUE SARCOMAS

Objective: Radiotherapy is essential for local control of soft tissue sarcomas (STS), yet the mechanisms underlying relapse within the irradiated field remain unclear. While DNA methylation is a key regulator of gene expression, the epigenetic evolution of STS across treatment and relapse is poorly understood. We sought to characterize methylation and transcriptional changes induced by radiotherapy to uncover epigenetic mechanisms driving radiation resistance.

Methods: We profiled 160 undifferentiated pleomorphic sarcoma and myxofibrosarcoma samples including primary, irradiated, recurrent tumors, and matched normals from 37 patients using RRBS and RNA-seq. RRBS data were processed with Bismark, and DMLs were identified with edgeR. RNA-seq data were quantified with Salmon and analyzed for differential expression using edgeR. HOMER was used to identify TF binding sites impacted by methylation changes. CNAs were inferred from exome sequencing using TitanCNA and integrated into PRISM to model subclonal methylation architecture.

Results: We identified over 130,000 DMLs across tumor progression. Primary and relapsed sarcomas showed predominant hypermethylation (~75%) in promoters, gene bodies, and intergenic regions, while irradiated tumors exhibited increased hypomethylation, suggesting therapy-induced disruption. In relapsed tumors, hypermethylation suppressed adhesion and transport pathways, whereas hypomethylation activated chromatin remodeling and ribosome biogenesis—supporting invasion, proliferation, and resistance. Integration with RNA-seq revealed silencing of tumor suppressors (e.g., CDKN2A) and activation of oncogenes (e.g., KIF5B, RRAS2). In irradiated tumors, hypomethylation-driven upregulation of CCND1 and DDB2 suggests enhanced DNA repair and cell-cycle bypass. Motif analysis implicated altered TF accessibility (e.g., SOX9, MAFB, PBX3), indicating regulatory rewiring. Subclonal analysis revealed increased epigenetic complexity and emergence of distinct methylation clones in relapse, consistent with clonal selection and resistance evolution.

Conclusion: These findings reveal the temporal and spatial dynamics of epigenetic remodeling in STS and nominate targets for relapse and radiation resistance. By uncovering methylation-driven regulatory changes and subclonal evolution, this work lays the groundwork for precision epigenetic therapies to overcome treatment resistance.