Abstract 1822: O-GlcNAcylation of NDRG1 mediates radiation resistance in prostate cancer

Prostate cancer (PCa) remains one of the most prevalent malignancies worldwide, with localized cases commonly treated through surgical resection and radiation therapy (RT). Both RT and androgen-targeted therapies can stimulate glutamine (L-Gln) secretion by the PCa-adjacent stroma, promoting tumor adaptation and resistance. Beyond its metabolic role, our findings revealed that L-Gln critically influences protein O-GlcNAcylation, a key process for DNA repair and therapy resistance. We hypothesized that targeting L-Gln signaling could enhance PCa sensitivity to RT. Our studies demonstrated that L-Gln starvation significantly increased RT sensitivity in both parental and radio-resistant PCa cell lines, a phenotype recapitulated by silencing OGT, the key enzyme mediating O-GlcNAcylation. Conditioned media from stromal fibroblasts elevated global O-GlcNAcylation and enhanced cell viability in PCa cells, highlighting the role of the tumor microenvironment. Sodium phenylbutyrate (SPB), a clinically available compound that sequesters L-Gln, effectively mimicked the effects of L-Gln depletion, contrasting with metabolic inhibitors of L-Gln that have failed to show clinical benefit. In subcutaneous xenograft models of PCa epithelia co-implanted with cancer-associated fibroblasts, the combination of SPB and RT markedly reduced tumor size compared to either treatment alone. Further, SPB significantly decreased plasma and intratumoral L-Gln concentrations. Mass spectrometry analysis of tumor tissues identified NDRG1 as a protein highly O-GlcNAcylated in response to RT, a modification suppressed by SPB. CRISPR/Cas9-mediated substitution of key O-GlcNAcylated serine residues in NDRG1 significantly enhanced RT sensitivity by destabilizing the protein and impairing its nuclear translocation. Mechanistically, NDRG1 dysfunction was associated with elevated endoplasmic reticulum (ER) stress, evidenced by increased CHOP and NRF2 expression and reduced Akt phosphorylation. RNA sequencing further revealed that SPB disrupted PCa cell metabolism by inhibiting fatty acid oxidation and reducing amino acid transporter activity, leading to diminished nucleotide pools and energy availability. This dual disruption of metabolic and O-GlcNAcylation processes compromised DNA repair and the detoxification of radiation-induced reactive oxygen species, thereby enhancing radiosensitivity. These findings suggest that targeting L-Gln bioavailability is a clinically viable strategy to overcome therapy resistance and improve therapeutic outcomes in PCa patients. Citation Format: Manish Thiruvalluvan, Sandrine Billet, Neil A. Bhowmick. O-GlcNAcylation of NDRG1 mediates radiation resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1822.