The incorporation of nucleoside analogs into DNA by polymerases, followed by their removal through base excision repair (BER), represents a promising strategy for cancer chemotherapy. In this study, we investigated the incorporation and cytotoxic effects of several nucleoside analogs—some of which are epigenetic reprogramming intermediates—in the U87 glioblastoma cell line. We found that two analogs, 5-hydroxymethyl-2′-deoxyuridine (5HmdU) and trifluorothymidine (TFT), are both cytotoxic and are efficiently incorporated into genomic DNA. In contrast, the 5-carboxy analogs—5-carboxy-2′-deoxyuridine (5CadU) and 5-carboxycytidine (5CadC)—showed no cytotoxicity and were not incorporated into DNA. Interestingly, 5-hydroxymethyl-2′-deoxycytidine (5HmdC) was cytotoxic but was not directly incorporated into DNA. Instead, it was deaminated into 5HmdU, which was then incorporated and likely responsible for the observed toxicity. 5HmdU is actively removed from DNA through the BER pathways. In contrast, TFT remains stably incorporated and is neither excised by BER nor does it hydrolyze into 5CadU—a known substrate for the DNA glycosylase SMUG1. We also found that N⁶-benzyladenosine (BzAdo), an inhibitor of the enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase (DNPH1), enhances the cytotoxicity of 5HmdU. However, the thymidine phosphorylase inhibitor tipiracil hydrochloride (TPI) does not increase the cytotoxic effect of TFT in U87 cells. Together, these findings highlight 5HmdU and TFT as promising chemotherapeutic agents for glioblastoma, each with distinct mechanisms of action and cellular processing.

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.

Expression levels of NQO1 and NAMPT across human tissue. Gene expression data of NQO1 and NAMPT across tissue samples from GTEx were compared. Dashed line represents the level of NQO1 and NAMPT expression in GBM for comparison.

Clonal assay of U87 and NHA cells treated with β-lap and FK866. Cells were treated with a titration of β-lap in the presence or absence of 10 nM FK866 in the same manner as in Figure 5D. Colonies were counted 2 weeks post exposure. A. U87 sensitivity by clonogenic assay. U87 cells are sensitive to β-lap and addition of FK866 increases this sensitivity. B. NHA sensitivity by clonogenic assay. NHA cells are resistant to β-lap exposure and addition of FK866 does not change NHA sensitivity to β-lap. Values are mean ± SD of n=3. *p<0.05, ****p<0.0001 between 0 and 10 nM FK866 groups. #p<0.05, ####p<0.0001 compared to untreated group (0 β-lap, 0 FK866). Statistical significance determined by one-way ANOVA with Sidak multiple comparison tests.

Targeting NQO1-expressing GBM with naphthoquinones. A, Western blot analysis demonstrating differential NQO1 expression across GBM cell lines. NQO1 expression was evaluated in NHAs, U87, and 6 PDX (GBM10, GBM39, GBM85, GBM117, GBM6, and GBM66) cell lines. Actin was used as the control. B, Quantification of Western blots. NQO1/actin expression was normalized to the reference U87 cell line. NQO1 expression is elevated in U87, GBM117, GBM10, and GBM39. C, Cartoon representation of reducible naphthoquinones including plumbagin, lapachol, and β-lap. Naphthoquinones may enter futile cycling by NQO1 reduction via NADH to generate reactive oxidative species (ROS). Accumulating ROS damage in DNA leads to PARP activation and NAD+ depletion. Severe ROS damage and NAD+ depletion lead to cell death. Qox and Qred represent the oxidized and reduced states of naphthoquinone substrates, respectively. Values are mean ± SD of n = 3. ANOVA and Dunnett test; **, P < 0.01; ****, P < 0.0001. [C, Created in BioRender. Chang-Gu, B. (2026) https://BioRender.com/jhhxbyp.]

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.