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Introduction/Objectives: Purines regulate DNA repair in brain tumors through uncertain mechanisms. We sought to define the mechanisms by which purines regulate DNA repair and therapy response. Methods: Crispr/Cas9, siRNA, and CDNA overexpression and immunoblot were used to modulate and confirm protein levels. γ-H2AX and Rad51 foci were enumerated using immunofluorescence. Fluorescent reporter assays were performed to detect DNA repair activity. Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after RT and antibodies generated against novel sites. Celltiter-glo or clonogenic assay were performed to evaluate drug/RT responses. IHC was used to detect protein expression. Animal models of glioblastoma and normal tissue (irradiation of GI system) were used to assess DNA repair and treatment responses in vivo. Results: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT, slowed the repair of RT-induced DSBs and inhibited the activity of non-homologous end joining (NHEJ), but not homologous recombination (HR). These effects could be rescued by GTP (but not ATP) supplementation. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced DSB repair and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to the small GTP-activated protein Rac1. Expression of constitutively active Rac1 promoted the dephosphorylation of Abi-1 (S323) and DSB repair while these effects were blocked by dominant negative Rac1. Pharmacogenomic inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. These findings have therapeutic relevance. In GBM PDX samples, levels of p-Abi-1 (S323) negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibition of Rac1 enhanced RT responses and suppressed Abi-1 S323 dephosphorylation. Abi-1 knockout enhanced efficacy of numerous genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in normal astrocytes and enteroids and protected mice from RT-mediated gastrointestinal injury. Conclusions: GTP promotes DNA repair by activating Rac1 and PP5 to dephosphorylate Abi-1 and stimulate NHEJ. Disrupting this regulation could improve brain tumor treatment responses while augmenting it could help protect normal tissues from genotoxic injury. Citation Format: Weihua Zhou, Angelica Lin, Zitong Zhao, John Yang, Kari Wilder-Romans, Jie Xu, Sumeet Solanki, Jing Li, Annabel Yang, Andrew Scott, Ayesha Kothari, Erik Peterson, Navyateja Korimerla, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra Obrien, Sean Ferris, Shuang Zhao, Jann N. Sarkaria, Meredith Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Yatrik Shah, Daniel R. Wahl. GTP-dependent signaling links metabolism, non-homologous end joining and treatment responses in cancer and normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3498.