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Background Radiation therapy (RT) activates nucleic acid sensing pathways and induces type I interferon (IFN-I) responses in cancer cells. However, glioblastoma remains refractory to RT and immunotherapy, reflecting potent mechanisms of immune evasion. We previously showed that RT induces a metabolic shift toward fatty acid synthase (FASN)-mediated lipid synthesis in glioblastoma. Since fatty acid metabolism can suppress IFN-I in viral immunity, we hypothesized that FASN induction in irradiated glioblastoma impairs IFN-I signaling and anti-tumor immunity.Methods In vitro, FASN was blocked using CRISPR/Cas9, inducible shRNA, or pharmacologic FASN inhibitors (FASNi) in murine glioblastoma cells (GL261, CT2A, SB28). Nucleic acid sensors (cGAS, RIG-I, and MDA5) were CRISPR/Cas9-deleted. Cells were irradiated (6Gy) and IFN-I assessed via RNA sequencing (RNAseq), LEGENDplex, and ELISA. A pan-cancer analysis using TCGA datasets assessed correlations between FASN expression and hallmark gene set enrichment scores across tumor types. In vivo, GL261 cells were orthotopically implanted and treated with fractionated RT (5×6Gy), FASNi, and/or anti-PD-1. CD8 or IFNAR blocking antibodies were used to determine the contribution of T cells and IFN-I signaling. Multiplex imaging using the CellScape™ platform (Bruker Spatial Biology) with an 11-marker panel assessed immune infiltration and was analyzed with Phenoplex (Visiopharm), which applies machine learning to classify immune cells and map their spatial distribution. Long-term survivors (>100 days) were rechallenged in the contralateral hemisphere.Results Unsupervised transcriptome analysis of irradiated FASN-knockout cells identified IFN-I signaling as the most enriched pathway, with corresponding increases in IFN-β and CXCL10 protein secretion. These responses were abrogated by deletion of cGAS, RIG-I, or MDA5. Importantly, TCGA pan-cancer analysis revealed that FASN expression inversely correlated with IFN and inflammatory response pathways in 32 tumor types. In vivo, RT or RT+FASNi similarly induced a marked increase in CD45+, CD8+, CD11b+, and CD11c+ cell infiltration in both the tumor core and margins. While adding anti-PD-1 or FASNi to RT failed to improve survival, the triple combination (RT+FASNi+anti-PD-1) resulted in 86% long-term survival versus 43% in other RT-treated groups. Rechallenged RT+FASNi+anti-PD-1 survivors remained tumor-free vs. naïve animals, demonstrating the induction of immunological memory. The therapeutic benefit was abolished by CD8 or IFNAR blockade.Conclusions FASN-driven lipid metabolism suppresses IFN-I responses in irradiated glioblastoma, enabling immune escape. Cross-cancer analysis suggests that FASN acts as a broad metabolic regulator of anti-tumor immunity. Critically, the combination of RT+FASNi+anti-PD-1 induces durable tumor rejection and immunological memory, highlighting FASN as a therapeutic target to sensitize irradiated glioblastoma to immunotherapy.Ethics Approval Animal studies were approved by the IACUC #2019-0042.