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Glioblastoma (GB) is a highly aggressive and therapy-resistant primary brain tumor with a dismal prognosis. Relapse typically occurs near the resection cavity, suggesting the local peritumoral area as its origin. GB cells from this region have been understudied and validated models are currently unavailable. Here, we analyzed matched tissue samples from the bulk tumor and the local peritumoral region of 11 GB patients, with samples from six patients sustaining growth beyond passage 6. Edge cultures were consistently more invasive but had lower self-renewal and tumorigenic capabilities compared to their matched bulk cultures. Whole exome sequencing (WES) analysis showed that edge cells were evolutionary early. Single nucleus (sn) RNA and ATAC-sequencing revealed a shift from less to more differentiated cell states in edge cultures. Cluster analysis identified core-like and edge-like clusters that could be further divided into three subclusters through snATAC-seq analysis. The bulk cell subclusters resembled known GB molecular subtypes, while two edge subclusters exhibited unique immune-related features. Our findings show that GB edge cells are functionally and molecularly distinct from their matched bulk cells and suggest that epigenomic immunoediting drives their progression, warranting extended investigations to understand their unique targets and vulnerabilities.

Glioblastoma (GB) is a highly malignant brain tumor characterized by significant heterogeneity, poor prognosis, and resistance to standard-of-care therapies. Using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) we identified two epigenetically distinct subgroups (ATAC-C2 and ATAC-C3) in IDH wild-type GB patients, revealing marked survival differences (median survival: 323.5 vs. 493 days). ATAC-C2 tumors showed higher treatment resistance, likely driven by mutant TP53 genotypes, while ATAC-C3 tumors exhibited intact p53 signaling and better responses to therapy. Functional analyses confirmed a predominance of dysfunctional TP53 mutations in ATAC-C2 and wild-type TP53 in ATAC-C3. In vitro studies demonstrated that p53 reactivation via PRIMA-1 treatment rescues viability in ATAC-C2 cell lines, but its efficacy was mutation-dependent. Connectivity Map analysis identified MG-132, a proteasome inhibitor, and SIB-1893, a selective antagonist of mGluR5, as potential therapeutic candidates for ATAC-C2. However, these compounds effectively reduced cell viability in both ATAC-C2 and ATAC-C3 cultures, suggesting a broader cytotoxic effect independent of p53 status. This study underscores the role of p53 and epigenetics in GB heterogeneity, survival, and treatment response and highlights the potential of adjuvant mutation-specific and novel agents for GB patients of the highly treatment-resistant ATAC-C2 subgroup.

A detailed understanding of epigenetic regulation in glioblastoma (GB) is crucial for identifying molecular mechanisms and developing targeted therapies for GB. To elucidate these mechanisms, we integrated analyses of chromatin accessibility, histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K27me3), and chromatin loops in human and mouse GCCs. Our analysis reveals that the enhancer marker H3K4me1 and the repressive marker H3K27me3 in human GCCs can separate patients into two subgroups with significant survival differences and enhancer signatures defining GB-subgroups. Trans-acting transcription factor (TF) enrichment analysis suggested that neural progenitor lineage-specific TFs, such as OLIG2, SOX4, POU family TFs (POU3F1, POU4F1, and POU3F4), SOX15, and FOXC2, are multiple acting TFs in different types of enhancers and determine the lineage specificity of human GCCs. Cross-species analysis between human and mouse GCCs identified key TFs that define lineage-specific subgroups and observed both conserved and species-specific regulatory mechanisms. Our work provides a comprehensive resource, enhances our understanding of the epigenetic landscape, and identifies potential targets for GB therapeutic intervention.