Abstract
BACKGROUND: Pathways that regulate differential energy metabolism in gliomas have recently emerged as promising therapeutic targets. Nicotinamide Phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway regulates key metabolic processes preferentially used in glioma energy metabolism and essential for tumor cell biology and proliferation. This study examines the effects of NAMPT inhibition using KPT9274 and FK866 (NAMPTi) on cellular respiration, oxidative stress and cytotoxicity to better delineate the role and regulation of NAD salvage pathway in gliomas. METHODS: Effects of NAMPTi on glycolysis and mitochondrial stress in gliomas and glioma stem cells (GSC) with varying IDH and MGMT status were measured including their oxidative state, basal cell respiration rate, maximum respiration capacity, spare respiratory capacity and proton leak using Agilent-Seahorse assay. Untargeted metabolomics was performed to analyze the effect of NAMPi on glioma cell metabolism. Effect of mitochondria dysfunction on cytotoxicity was measured by annexin-PI and CaspaseGlo assays. RESULTS: NAMPT inhibition caused NAD depletion, reduced ATP levels and PAK4 downregulation in gliomas. NAMPTi-treated cells showed reduction in basal cell respiration, spare and maximum respiration capacity indicating mitochondrial dysfunction and oxidative stress in glioma cells resulting in programed cell death. Untargeted metabolomics study indicated specific metabolic changes including accumulation of oxidized glutathione in NAMPTi-treated cells, indicating oxidative stress. Further, accumulation of metabolites indicative of a glycolytic pathway block and shunt towards the de novo purine synthesis pathway was seen. Further, glycolysis inhibition by NAMPTi was confirmed by glycolysis stress assays. Lastly, glycolysis inhibition and mitostress appeared to be related to a decrease in NAD+ dependent SIRT1 expression. CONCLUSIONS: NAMPTi cause profound disruption of mitochondrial function, induce oxidative stress and trigger cytotoxicity in GSC irrespective of MGMT promoter-methylation or IDH1 status. Targeting NAMPT is hence a novel therapeutic strategy that potentially circumvents tumor heterogeneity and can specifically disables tumor cell metabolism.