Abstract
Background: Glioblastoma (GBM) exhibits profound intratumoral heterogeneity and a high propensity for developing drug tolerance, yet the chromatin-level mechanisms driving this adaptation remain poorly understood. Single-cell ATAC-seq enables unbiased profiling of chromatin accessibility at unprecedented resolution, offering a window into epigenetic reprogramming during drug exposure. Methods: We performed single-cell ATAC-seq on patient-derived GBM cells treated with temozolomide (TMZ) over a 14-day time course. Computational analysis identified distinct chromatin states associated with drug-tolerant versus sensitive subpopulations, and transcription factor motif enrichment was assessed. Integrated comparisons with matched single-cell RNA-seq data were conducted. Results: We profiled 12,450 cells across five time points and identified a drug-tolerant cell state characterized by increased chromatin accessibility at loci regulating stemness, DNA repair, and anti-apoptotic pathways. Motif analysis revealed enrichment of SOX, POU, and FOX family transcription factors in tolerant cells. Dynamic shifts in accessibility were observed within 48 hours of treatment and became fixed by day 7, suggesting an ordered epigenetic cascade. Integrative analysis linked accessible regions to genes involved in glutathione metabolism and Notch signaling. Conclusions: Our results reveal that drug tolerance in GBM is accompanied by a rapid and coordinated reprogramming of chromatin accessibility, driven by specific transcription factor networks. These findings highlight the potential of targeting epigenetic plasticity to overcome therapy resistance in glioblastoma.