41 Pharmacological depletion of NAD+ has single agent cytotoxicity and radiosensitises glioma stem-like cells through impaired DNA replication and repair

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Abstract Introduction Metabolic rewiring contributes to radioresistance and poor outcomes in glioblastoma. Nicotinamide phosphoribosyltransferase (NAMPT) is rate-limiting in the NAD salvage pathway and overexpressed in GBM; high expression correlates with poor survival. We hypothesised that the NAMPT inhibitor FK866 would overcome radioresistance in GBM models. Methods Effects on proliferation, clonogenic survival, cell cycle, gene expression and DNA damage responses were measured in genetically diverse, patient-derived glioblastoma stem-like cells (GSCs). Toxicity was assessed in CD1 nude mice treated with FK866 +/- brain irradiation. Results FK866 exhibited potent cytotoxicity in sixteen GSC lines (IC50 0.25-13.84nM) irrespective of genetic background, MGMT status or baseline NAMPT expression. Normal human astrocytes were unaffected. Supplementation with NAD or nicotinamide mononucleotide reversed cytotoxic effects, validating NAMPT inhibition and NAD depletion as mechanisms. FK866 rapidly depleted NAD, causing ATP reduction and progressive gene expression changes. However, drug removal after 72 hours permitted recovery and proliferation, indicating the need for combination approaches. Clonogenic and spheroid growth delay assays showed that low nanomolar concentrations of FK866 significantly enhanced radiosensitivity in multiple GBM cell lines. Mechanistically, NAMPT inhibition perturbed energy and nucleotide metabolism, disrupting DNA replication and inhibiting cell-cycle progression. Prolonged exposure to FK866 suppressed ATR–CHK1 signalling and reduced RAD51 expression alongside downregulation of replication/repair transcripts. Accordingly, FK866 impaired resolution of radiation-induced γH2AX foci. In vivo, FK866 (15 and 30 mg/kg), alone and in combination with brain irradiation (6 x 2 Gy), was well tolerated by non-tumour bearing CD1 nude mice with no weight loss or neurological signs observed. Conclusions Targeting NAMPT with FK866 has therapeutic potential in glioblastoma both as a single agent and a potent radiosensitiser. The absence of overt toxicity supports further investigation in orthotopic efficacy studies.

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