Nuclear cholesterol regulates nuclear size and DNA damage responses in cancer stem cells

Abstract Background Nuclear atypia is associated with increased malignancy in numerous cancers, including glioblastoma (GBM). Here, we found that GBM stem cells display small nuclear size, prompting investigation of mechanisms underlying nuclear size regulation in the tumor hierarchy. Methods We performed comparative gene expression and proteomics in GBM stem cells (GSCs) and neural stem cells (NSCs) to discover potential regulators of nuclear size. Through transcriptomic analysis, mass spectrometry, and pharmacologic inhibition, we interrogated the functional significance of nuclear size regulation. Results GSCs were enriched for a nuclear sterol reductase, Lamin B Receptor (LBR). Targeting LBR increased nuclear size and decreased GSC viability and tumor initiation. Regulation of nuclear cholesterol synthesis underlaid LBR-dependency in GSCs. Loss of LBR or reduction of cholesterol levels induced double-strand DNA breaks (DSBs), activating P53-dependent DNA damage responses (DDR). The GSC proteomic LBR interactome revealed DDR mediators, including DEAD-box RNA helicase DDX5 that resolves R-loops at DSBs. Genetic targeting of LBR reduced the DDX5-R loop interaction, leading to increased R-loop formation rescued by cholesterol supplementation. Pharmacological sterol reductase inhibition mirrored genetic LBR targeting by reducing the DDX5-R loop interaction and increasing R-loops and DSBs. Targeting LBR genetically and pharmacologically inhibits GSC growth in vivo and synergizes with irradiation. Conclusions Stem-like GBM cells display reduced nuclear size, driven by nuclear cholesterol synthesis to regulate radiation responses, revealing a novel therapeutic paradigm.