Integrated genetic screening identifies FEN1 as a selective vulnerability and temozolomide resistance mediator in glioblastoma stem cells.

Glioblastoma (GBM) is an aggressive brain tumor characterized by therapy resistance and recurrence. Glioblastoma stem cells (GSCs) are key drivers of tumor maintenance, therapeutic resistance, and relapse, but targeting them remains clinically elusive due to their overlap with normal neural stem cells (NSCs) and a lack of actionable vulnerabilities. To identify selective vulnerabilities in GSCs, we performed genome-wide CRISPR-Cas9 loss-of-function screening across patient-derived GSC models under standard-of-care treatment conditions. We identified flap endonuclease 1 (FEN1), a key enzyme in DNA replication and base excision repair, as an essential gene for GSC survival, with enhanced dependency in the context of temozolomide (TMZ) treatment. Genetic knockdown of FEN1 impaired GSC proliferation and self-renewal and extended survival in a patient-derived xenograft model. Pharmacologic inhibition of FEN1 using a small-molecule inhibitor revealed selective cytotoxicity in highly aggressive and recurrent GBM models, while sparing NSCs. Notably, FEN1 inhibition synergized with TMZ to induce DNA double-strand breaks and potentiate cell death only in a subset of GSCs sensitive to FEN1 inhibition. Mechanistically, single-cell transcriptomics revealed that FEN1 expression correlates with programs linked to proliferation, stemness, and DNA damage repair, underscoring its role in maintaining the treatment-refractory phenotype. Our findings identify FEN1 as a selective vulnerability in aggressive, proliferative GSCs. FEN1 inhibition not only impairs GSC viability but also restores sensitivity to TMZ in treatment-resistant models, offering a strategy for salvage therapy in recurrent GBM. These results support the development of FEN1-targeted therapies and lay the foundation for a biomarker-guided approach to overcome chemoresistance in GBM.