Abstract
Glioblastoma multiforme (GBM) represents the most aggressive and lethal primary brain malignancy in adults, characterized by profound epigenetic dysregulation and extensive transcriptional heterogeneity. While genomic drivers of GBM have been comprehensively cataloged, the non-coding regulatory landscape governing oncogenic transcriptional networks remains largely uncharted. In this study, we coupled high-throughput computational epigenomic profiling with targeted CRISPR-dCas9-mediated epigenome editing to systematically identify and functionally validate novel cis-regulatory elements (CREs) implicated in GBM pathogenesis. By integrating ATAC-seq, ChIP-seq for active enhancer marks (H3K27ac and H3K4me1), and Hi-C chromatin conformation data across primary patient-derived GBM stem cells, we computationally prioritized 48 candidate enhancer loci. We subsequently deployed a pooled dCas9-KRAB repressive screening approach, followed by targeted dCas9-p300 activation, to perturb the chromatin architecture of these non-coding elements. Our functional dissection uncovered a previously uncharacterized distal super-enhancer cluster, designated Enh-GBM-17, situated 85 kilobases upstream of the SOX2 locus, which directly orchestrates stemness and temozolomide resistance via long-range chromatin looping. Epigenetic silencing of Enh-GBM-17 led to robust downregulation of SOX2, impaired sphere formation, and attenuated tumorigenicity in orthotopic xenograft models. These findings delineate the essential role of distal regulomes in driving GBM progression and demonstrate the power of integrative functional epigenomics to unveil therapeutic vulnerabilities hidden within the dark matter of the cancer genome.
