EPCO-25. SINGLE-NUCLEUS CO-PROFILING OF TRANSCRIPTOME, HISTONE MODIFICATIONS, AND CHROMATIN ACCESSIBILITY REVEALS DYNAMIC EVOLUTION OF RECURRENT GLIOBLASTOMA

Abstract Recent breakthroughs in genome technologies such Paired-Seq and Pair-Tag can jointly profile transcriptome and chromatin accessibility or histone modifications in single cells, and these methods have been used to study cell-type specific regulatory networks in normal human brains. However this approach has not yet been applied to any complex disease or primary tumor. Here, for the first time, we performed single-nucleus paired-tag characterization of freshly resected tissues from three recurrent glioblastoma (GBM) patients, including tumor core and adjacent brain specimens, to understand the epigenetic mechanisms that facilitate GBM cell aggressiveness and evolution. Clustering of over 25,000 cells across modalities identified distinctive normal and neoplastic populations, the latter of which exhibited clonal copy number amplifications in established oncogenes such as EGFR, MDM2, and PDGFRA. Cell states were sharply defined by unique enhancer profiles, while repressive regions existed in a broader continuum with overlap of glial, neuronal, and tumor populations. Detection of cell-type specific H3K27ac and H3K27me3 peaks revealed lineage restricted cis regulatory elements (CRE) that strongly correlated with gene expression patterns, including potent enhancers and silencers associated with cell cycle regulation. Co-accessibility analyses of CREs with nearby promotors further elucidated transcriptional mediators, such as tumor-specific enhancers of the glioma invasion gene HMGA2. Stratification of GBM cells into previously established transcriptional states (MES, NPC, OPC, AC) identified key epigenetic facilitators associated with subgroup-specific expression patterns, as well as downstream evolutionary trajectories that drive clonal selection and therapy resistance. Our results leverage granular co-profiling of histone modifications, gene expression, and chromatin organization to derive novel insights into recurrent GBM biology, identifying new therapeutic vulnerabilities in these molecularly divergent lesions.