41 Elucidating the altered metabolism of NAD+ in the selective targeting of glioblastoma with the NQO1- activated drug β-lapachone

Abstract

Objectives/Goals: NAD+ synthesis is enhanced in glioblastoma (GBM) allowing GBM to resist chemotherapy. NQO1 is upregulated in GBM and may be targeted by β-lapachone (β-lap) to induce NAD-depletion and cell death. This project investigates NQO1 as a selective target for GBM and the contributions of glucose and uridine to NAD+ synthesis. Methods/Study Population: Survival Studies and NQO1 expression. RNA-seq and survival data from TCGA of glioma patients (n = 667) was obtained using the UCSC Xena platform. Western blots were utilized to determine expression levels of NQO1 and NAMPT in normal human astrocytes, U87 cells, and patient-derived GBM cell lines. Immunocytochemistry: γ-H2AX staining was used to evaluate β-lap induced DNA damage. NQO1-dependence was evaluated with the NQO1-inhibitor dicoumarol. Cytotoxicity measurements. Cells were exposed to β-lap and other inhibitors, and cell survival was determined by trypan-blue exclusion assay. Co-culturing experiments were performed with fluorescently labeled U87 cells and unlabeled astrocytes. NAD+ quantification. Intracellular NAD+ was acid extracted and quantified by an enzyme-cycling reaction. Results/Anticipated Results: NQO1 overexpression is linked to decreased survival in glioma patients. In glioma patients, high NQO1 expression was associated with a decreased overall survival and high-grade tumors. β-lap induces selective NAD+ depletion and cell death in NQO1-expressing GBM cells. Western blots demonstrate NQO1 expression to be elevated in GBM cell lines compared to normal human astrocytes. β-lap induces NQO1-dependent NAD+ depletion and cell death in GBM compared to astrocytes in mono- and co-culture experiments. Glucose and uridine facilitate NAD+ regeneration in GBM. We demonstrate extracellular glucose and uridine facilitate NAD+ regeneration and cell survival in β-lap exposed GBM cells. Utilizing inhibitors, we determined that glucose and uridine facilitate NAD+ regeneration through the NAD+ salvage pathway. Discussion/Significance of Impact: GBM is the most common primary adult CNS tumor with a median survival of 14 months. Despite significant research in therapeutic strategies, treatment has not improved in 2 decades. There is a significant need to discover new targets that may improve GBM treatment. We demonstrate here that targeting NQO1 with β-lap induces selective GBM toxicity.