Abstract
Glioblastoma (GBM) is a highly aggressive brain tumor with limited response to standard of care chemoradiotherapy. In this study, we conducted genome-wide CRISPR knockout screening in patient-derived glioblastoma stem cells (GSCs) to identify genetic dependencies of cell survival and therapy resistance. Our screening identified flap endonuclease 1 (FEN1) as a key driver of GSC survival, with enhanced dependency under temozolomide (TMZ) treatment. Genetic perturbation of FEN1 reduced GSC self-renewal and proliferation in vitro, and prolonged survival in a patient-derived xenograft model of GBM. FEN1 inhibition preferentially impacted highly aggressive or recurrent GBM models compared with less aggressive GBMs and healthy neural stem cells (NSCs). Promisingly, FEN1 inhibition synergized with TMZ only in these high-FEN1-dependency GSCs, providing cancer-selective killing and TMZ sensitization in the most untreatable of GBMs. Mechanistically, high-FEN1-dependency GSCs displayed greater proliferation and sphere formation, while reducing their proliferation conferred resistance to FEN1 inhibition. To more broadly characterize the determinants of FEN1 dependency, bulk and single cell transcriptomics linked FEN1 expression to stemness and proliferation, while low-FEN1-dependency GSCs exhibited upregulation of neuronal differentiation pathways. These results establish FEN1 as a cancer-selective and TMZ-sensitizing therapeutic target in GBM, highlighting its potential for therapeutic exploitation in aggressive cancers.