Abstract Background: Prostate cancer (PCa) frequently develops resistance to radiation therapy (RT), driven in part by DNA repair mechanisms. We previously found that glutamine (L-Gln) enables protein O-GlcNAcylation post-translational modifications linked to DNA repair and therapy resistance. This is highly relevant to irradiation of PCa metastatic sites like the bone and liver microenvironments that have markedly high concentrations of L-Gln and accordingly poor response to irradiation. Here, we investigated whether pharmacologic glutamine depletion using sodium phenylbutyrate (SPB) disrupts this axis and enhances radiosensitivity. Methods: Radio-resistant 22Rv1 and ARCaPM lines were generated by iterative chronic irradiation. Subcutaneous and liver xenografts were treated with vehicle, SPB, RT, or SPB+RT. Tumors analyzed by bulk RNA-seq, proteomics, and immunoblotting revealed the importance of NDRG1 and PRDX1 o-glycosylation. CRISPR/Cas9 mutagenesis was used to generate O-GlcNAc-deficient NDRG1 and PRDX1 variants. DNA-damage repair, cell-cycle dynamics, and mitochondrial function were assessed by γH2AX staining, flow cytometry, and Seahorse assays. Results: SPB+RT produced the greatest tumor reduction and significantly reduced circulating and intratumoral glutamine, compared to either RT or SBP alone. Transcriptomic and proteomic analyses showed downregulation of amino-acid transport, fatty-acid metabolism, and histone demethylase activity, indicating broad metabolic and epigenetic reprogramming. Mass spectrometry identified NDRG1 and PRDX1 as radiation-induced O-GlcNAc targets suppressed by SPB. CRISPR-engineered O-GlcNAc-deficient NDRG1 and PRDX1 variants exhibited greater RT sensitivity as a result of persistent γH2AX foci, prolonged G2/M arrest, reduced nuclear localization, and decreased protein stability. RNA-seq of these variants showed enrichment of p53 signaling, endoplasmic reticular (ER) stress with metabolic compensation through increased c-Myc activity, and oxidative phosphorylation. Nucleoside supplementation did not reverse SPB-mediated radio-sensitization, indicating that SPB’s effects extended beyond nucleotide depletion. Instead, the data suggested the role of L-Gln addiction in irradiated PCa tumors was due to NDRG1 and PRDX1 in ER stress-response proteins and activating the unfolded protein response. Conclusions: O-GlcNAcylation of NDRG1 and PRDX1 stabilizes stress-response proteins to support DNA-repair and metabolic fitness after irradiation. SPB disrupts this L-Gln-driven O-GlcNAcylation axis, to impair protein translation supporting significant radio-sensitization. As SPB is used for chronic management of urea cycle disorders, repurposing to overcome radiation resistance provides a near-term therapeutic translation opportunity for PCa patients. Citation Format: Manish Thiruvalluvan, Sandrine Billet, Saravana Kumar Kailasam Mani, Joshua Watson, Neil A. Bhowmick, . Limiting O-GlcNAcylation support prostate cancer radiation sensitivity through metabolic and epigenetic reprogramming abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6616.
Thiruvalluvan et al. (Fri,) studied this question.