Xujia Wu, Donghai Wang, Suchet Taori, Weichi Wu, Deobrat Dixit, Deguan Lv, Steven J. Mullett, Stacy L. Gelhaus, Fanen Yuan, Po Zhang, Tengfei Huang, Huairui Yuan, Qiulian Wu, Jeremy N. Rich
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 N 4 -acetylcytidine (ac 4 C) 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/ac 4 C-modified R-loops using remodelin phenocopies NAT10 genetic targeting, demonstrating therapeutic promise for targeting cancer.