Abstract 6799: Dissecting the epigenetic inter- and intra-tumor heterogeneity of glioma at single-cell resolution

Abstract Adult diffuse gliomas include IDH-mutant astrocytomas and oligodendrogliomas, as well as IDH-wildtype glioblastomas (GBM), the latter marked by extreme cellular plasticity. Bulk genomic studies from TCGA defined molecular subtypes, but how epigenetic programs coordinate state transitions within tumors remains unresolved. In this study, we applied single-nucleus methylome and 3D genome profiling (snm3C-seq) to 40 human gliomas and interrogated the heterogeneity of DNA methylation and chromatin structure within tumor and across donors. We assigned DNA methylation and chromatin conformation (Hi-C) cell states to individual cells. We reported the following results: 1) Cross-modal state mapping: Most tumors contain large fractions of OPC-like or Astro-like cells with strong concordance between methylation (mC) and Hi-C states. However, certain clusters of cells have discordant mC and Hi-C states, indicating transitional phenotypes. 2) Temporal discrepancy: We observe asynchronous remodeling, where chromatin architecture often shifts towards more differentiated states before DNA methylation during lineage transitions. For example, many cells are OPC-like in methylation and AC-like in chromatin conformation, suggesting 3D genome reorganization as an early indicator of fate change. 3) Copy number alterations in tumor associated normal cells: we detected CNV mosaicism in a large proportion of non-malignant cells, yet their methylation profiles are more similar to normal cells than cancer cells. 4) Clonal evolution and structural variants (SV): Clonal analysis by copy number reveals that some new malignant clones arise with global methylation loss. In contrast to the heterogenous copy number changes in cancer cells, structural variants are often shared by the majority of cancer cell proportions. However, the occurrence of new SV drives cell state transition more strongly than copy number changes. Lastly, we observed that extrachromosomal DNA (ecDNA) are frequently found in WT GBM and they are associated with local epigenetic rewiring at amplified loci. These insights nominate epigenetic states as a biomarker of plasticity and a potential guide for patient-specific interventions. Citation Format: Yue Wu, Jingtian Zhou, Zhiyi Deng, Kaushik Komandur, King L. Hung, Qiurui Zheng, Anna bartlett, Joseph Nery, Rosa Gomez Castanon, Thomas Beaumont, Jesse Dixon, Joseph R. Ecker. Dissecting the epigenetic inter- and intra-tumor heterogeneity of glioma at single-cell resolution abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6799.