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. Here, 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) were evaluated. Dose-response studies, NAD+ quantification, and immunoblot were utilized to evaluate the link between NAD+ synthesis and β-lap toxicity. We demonstrate 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 NHA. 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 targeting NQO1-expressing GBM with NQO1 bioactivatable compounds in combination with NAMPT-inhibitors, is a promising therapeutic strategy for the treatment of GBM.