Targeting the NAD+ Salvage Pathway Blocks Metabolic Recovery and Enhances β-Lapachone Toxicity in NQO1-Expressing Glioblastoma Cells.

Glioblastoma (GBM) is a highly heterogeneous tumor, with some cell populations overexpressing genes that give them a survival advantage. One such gene is NAD(P)H quinone oxidoreductase 1 (NQO1), which protects cells from oxidative stress. Although NQO1 overexpression shields cells from endogenous oxidants, the enzyme can also bioactivate certain compounds, including the naphthoquinone β-lapachone (β-lap). This activation triggers repeated cycles of oxidative stress that ultimately drive cell death. In this study, we investigated whether NQO1 overexpression could be exploited as a selective vulnerability to induce toxicity in GBM cells. NQO1 expression status in the U87 GBM cell line, multiple patient-derived GBM cell lines, and normal human astrocytes (NHA) was evaluated. Dose-response studies, NAD+ quantification, and immunoblot analysis were utilized to evaluate the link between NAD+ synthesis and β-lap toxicity. We demonstrate that NQO1 is highly expressed in multiple GBM cell lines and β-lap induces selective cytotoxicity in these cells while sparing low NQO1-expressing cells including NHAs. β-lap induces acute NAD+ depletion and DNA damage. However, high NQO1-expressing GBM cells may regenerate NAD+ and evade β-lap toxicity. We identified the NAD+ salvage pathway to be the primary pathway responsible for maintaining NAD+ levels in GBM. We demonstrate that targeting this pathway with the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 prevents NAD+ regeneration after β-lap exposure and enhances β-lap cytotoxicity in NQO1-expressing GBM. Altered NAD+ metabolism in GBM represents a potential metabolic vulnerability. Our results suggest that targeting NQO1-expressing GBM with NQO1-bioactivatable compounds in combination with NAMPT inhibitors is a promising therapeutic strategy for the treatment of GBM. SIGNIFICANCE: NAD+ metabolism is altered in GBM. However, there are limited therapeutic options for targeting this metabolic vulnerability. This study identifies NQO1+ GBM as highly susceptible to β-lap-induced NAD+ depletion and cell death. Blocking the NAD+ salvage pathway prevents metabolic recovery in GBM and increases β-lap toxicity in NQO1+ cells.