Electron transport chain inhibition increases cellular dependence on purine transport and salvage.

Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.

Electron transport chain inhibition increases cellular dependence on purine transport and salvage.

Cancer cells reprogram their metabolism to support cell growth and proliferation in harsh environments. While many studies have documented the importance of mitochondrial oxidative phosphorylation (OXPHOS) in tumor growth, some cancer cells experience conditions of reduced OXPHOS in vivo and induce alternative metabolic pathways to compensate. To assess how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts and plasma from patients with inborn errors of mitochondrial metabolism, and in cancer cells subjected to inhibition of the electron transport chain (ETC). All these analyses revealed extensive perturbations in purine-related metabolites; in non-small cell lung cancer (NSCLC) cells, ETC blockade led to purine metabolite accumulation arising from a reduced cytosolic NAD + /NADH ratio (NADH reductive stress). Stable isotope tracing demonstrated that ETC deficiency suppressed de novo purine nucleotide synthesis while enhancing purine salvage. Analysis of NSCLC patients infused with [U- 13 C]glucose revealed that tumors with markers of low oxidative mitochondrial metabolism exhibited high expression of the purine salvage enzyme HPRT1 and abundant levels of the HPRT1 product inosine monophosphate (IMP). ETC blockade also induced production of ribose-5' phosphate (R5P) by the pentose phosphate pathway (PPP) and import of purine nucleobases. Blocking either HPRT1 or nucleoside transporters sensitized cancer cells to ETC inhibition, and overexpressing nucleoside transporters was sufficient to drive growth of NSCLC xenografts. Collectively, this study mechanistically delineates how cells compensate for suppressed purine metabolism in response to ETC blockade, and uncovers a new metabolic vulnerability in tumors experiencing NADH excess.