EXTH-98. BRI3 Promotes Glioblastoma Progression and Radiation Resistance via GPX8-Driven Redox Homeostasis

Abstract BACKGROUND We previously identified Brain Protein I3 (BRI3) gene dosage gain as a robust prognostic biomarker in IDH-wildtype glioblastoma (GBM). However, the mechanism by which BRI3 contributes to radiation resistance and tumor progression remained unknown. We hypothesized that BRI3 promotes resistance through transcriptional regulation of redox homeostasis genes critical for DNA damage evasion. METHODS We developed three complementary CRISPR-based models: (1) a murine GBM cell line (CR86) with BRI3 knockout (KO), (2) a human GBM cell line (LN229-BRI3-KO), and (3) an in vivo murine GBM model with BRI3 KO induced via in utero electroporation. Functional characterization included proliferation assays, clonogenic survival post-radiation, RNA sequencing (pre-/post-RT), Seahorse metabolic flux analysis, and immunoblotting. Public transcriptomic datasets from Rembrandt, TCGA, and Gravendeel were intersected with curated lists of DNA damage response (DDR) and reactive oxygen species (ROS) scavenger genes to identify BRI3-regulated candidates. RESULTS BRI3 KO significantly sensitized CR86 cells to radiation across a dose range (p < 0.0001), with clonogenic survival reduced >3-fold at 8 Gy compared to BRI3-WT controls. Seahorse profiling revealed a shift toward oxidative phosphorylation and reduced glycolysis in KO cells, accompanied by suppression of glutathione-linked ROS detoxification. RNA-seq identified GPX8—a glutathione peroxidase—as the dominant downstream gene repressed in BRI3 KO cells across both irradiated and unirradiated conditions in murine and human models. Western blot analyses confirmed post-RT GPX8 upregulation in BRI3-WT and its near absence in KO lines. In vivo, BRI3 KO tumors exhibited markedly delayed progression (median survival >300 vs. <100 days), correlated with reduced phospho-AKT signaling, suggesting BRI3 may also promote tumor growth via AKT activation. CONCLUSIONS BRI3 facilitates glioblastoma progression and radiation resistance via GPX8-dependent ROS scavenging and AKT-mediated proliferation. These findings nominate the BRI3-GPX8 axis as a novel therapeutic vulnerability in GBM.