NAT10-dependent N⁴-acetylcytidine reprograms R-loops and promotes cancer stem cell growth

R-loop remodeling dynamically regulates chromatin states and gene expression; however, its exploitation by cancer to sustain self-renewal and malignancy remains poorly understood. Here, we find that glioblastoma (GBM) stem cells (GSCs) display highly active R-loops compared to differentiated progeny and neural stem cells. Genome-wide mapping reveals cell-specific enrichment and spatial accumulation of R-loops at promoter-proximal regions in GSCs, correlating with active transcription and open chromatin. We identify N-acetyltransferase 10 (NAT10) as a high-affinity R-loop-binding protein in GSCs, where it is overexpressed downstream of OLIG1. NAT10 catalyzes widespread N⁴-acetylcytidine (ac⁴C) deposition on the RNA strand of R-loops, stabilizing promoter-associated R-loops and facilitating open chromatin to sustain self-renewal through core stemness regulators, including EGR1. NAT10 knockdown suppresses GSC proliferation and maintenance in vitro and attenuates tumor growth in vivo. Pharmacological inhibition of NAT10/ac⁴C-modified R-loops using remodelin phenocopies NAT10 genetic targeting, demonstrating therapeutic promise for targeting cancer.