Abstract B040: 53BP1-T336 Phosphorylation by GSK3β regulates DNA damage response and radioresistance in GBM

Abstract Objectives: Glioblastoma multiform (GBM) is the most lethal primary brain tumor, with combination of surgery, radiation therapy (RT), and temozolomide (TMZ) as the standard of care. Despite treatment, over 50% of tumors recur within six months, and more than 70% of patients die within two years, highlighting profound radioresistance. This clinical radioresistance is partly attributed to intrinsic DNA damage-response pathways (DDR) within GBM cells and tumor microenvironment (TME) protecting GBM from RT cytotoxicity. We have previously established that glycogen synthase kinase 3β (GSK3β), highly expressed in the brain, phosphorylates 53BP1 at threonine 336 (T336), impeding its function in repairing RT-induced DNA double-strand breaks (DSBs). We aim to elucidate the role of the GSK3β-53BP1 axis in GBM progression and response to RT and other genotoxic treatment modalities. Methods: To better understand the role of 53BP1-T336 phosphorylation by GSK3β in GBM tumorigenesis and to uncouple the GSK3β-53BP1 axis in vivo, we generated a CRISPR/Cas9-mediated homozygous knock-in mutant mice with a 53BP1 T336A mutation, preventing GSK3β-mediated T336 phosphorylation. Utilizing syngeneic orthotopic glioma models derived from mouse glioma cells GL216 and SB28, we explored how systemic 53BP1-T336A mutation modulates glioma progression and radiosensitivity. RNA sequencing (RNA-seq) and proteomics analyses were employed to analyze gene expression changes in both the mouse glioma and its microenvironment. Furthermore, we evaluated the clinical relevance of altered genes in gliomas from 53BP1-T336A mice using the TCGA database. Results: Systemic ablation of the GSK3β/53BP1 axis in genetically engineered 53BP1-T336A mice suppressed glioma progression, prolonged mouse survival, and enhanced tumor response to RT. The uncovered T336A mutation-induced transcriptomic and proteomic profile alterations contribute to functional regulation during glioma progression. Tumors from T336A mice showed increased DNA damage accumulation, through elevated levels of the DNA damage marker γH2AX. Gene ontology (GO) analysis of overall differentially expressed genes (DEGs) with revealed that systemic T336A mutation upregulated neuronal repair and immune function pathways while downregulating pathways contributing to invasion and tumor metastasis, a findings supported by transcriptomic and proteomic data. Notably, T336A mutation-associated differentially expressed genes correlated with glioma patient overall survival in the TCGA database using Kaplan–Meier survival curves along with the log-rank test (human genome atlas data). Conclusion: Our investigations unveiled a novel role of the host GSK3β/53BP1 axis in regulating glioma progression and response to RT, providing an unbiased data source for understanding 53BP1T336A-induced transcriptomic and proteomic alterations in glioma. Citation Format: Heba Allam, Grayson Young, Fen Xia. 53BP1-T336 Phosphorylation by GSK3β regulates DNA damage response and radioresistance in GBM [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B040.