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.

De novo and salvage purine synthesis pathways across tissues and tumors.

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.

Adipocytes reprogram cancer cell metabolism by diverting glucose towards glycerol-3-phosphate thereby promoting metastasis.

In the tumor microenvironment, adipocytes function as an alternate fuel source for cancer cells. However, whether adipocytes influence macromolecular biosynthesis in cancer cells is unknown. Here we systematically characterized the bidirectional interaction between primary human adipocytes and ovarian cancer (OvCa) cells using multi-platform metabolomics, imaging mass spectrometry, isotope tracing and gene expression analysis. We report that, in OvCa cells co-cultured with adipocytes and in metastatic tumors, a part of the glucose from glycolysis is utilized for the biosynthesis of glycerol-3-phosphate (G3P). Normoxic HIF1α protein regulates the altered flow of glucose-derived carbons in cancer cells, resulting in increased glycerophospholipids and triacylglycerol synthesis. The knockdown of HIF1α or G3P acyltransferase 3 (a regulatory enzyme of glycerophospholipid synthesis) reduced metastasis in xenograft models of OvCa. In summary, we show that, in an adipose-rich tumor microenvironment, cancer cells generate G3P as a precursor for critical membrane and signaling components, thereby promoting metastasis. Targeting biosynthetic processes specific to adipose-rich tumor microenvironments might be an effective strategy against metastasis.

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.

Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

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.

L-2-hydroxyglutaric aciduria - review of literature and case series.

L-2-hydroxyglutaric aciduria (L2HGA) is an autosomal recessive, slowly progressive neurodegenerative disease characterized by psychomotor delay and cerebellar dysfunction. The biochemical hallmark is increased concentrations of L2HG in body fluids. Brain MRI exhibits characteristic centripetal extension of the white matter involvement that differentiates it from other leukodystrophies. The authors report two sisters from Pakistan with L2HGA with 4 years of follow-up. The authors have also compared the clinical outcome of our patients with 45 previously reported patients with L2HGA for whom treatment and clinical outcome was reported. Case presentation: The authors report two sisters with L2HGA from Pakistan born to consanguineous parents. The 15- and 17-year-old girls presented with psychomotor delay, seizures, ataxia, intentional tremors, and dysarthria. Both had normal anthropometric measurements for age. Exaggerated tendon reflexes and bilateral sustained ankle clonus were observed in addition to cerebellar signs. Urine organic acids analysis showed marked excretion of 2-hydroxyglutaric acid, chiral differentiation of 2-hydroxyglutaric acid showed it to be L2HGA. Brain MRI of the 15-year-old showed diffuse subcortical white matter changes evident by T2/FLAIR hyperintense signals bilaterally, particularly in the frontal region in the centripetal distribution with some diffusion restriction along involvement of globus pallidus. The characteristic MRI pattern raised the suspicion of L2HGA. Targeted L2HGDH sequencing identified a homozygous pathogenic variant, c.829C>T (p.Arg227*) in L2HGDH gene in both girls. Both parents were heterozygous carriers of the familial variant. Conclusion: Neuroradiological features of centripetal subcortical leukoencephalopathy with basal ganglia and dentate nuclei involvement are rather specific to L2HGA and should lead to further biochemical investigations to look for L2HGA and L2HGDH gene sequencing.

Mitochondrial metabolism in primary and metastatic human kidney cancers.

Most kidney cancers display evidence of metabolic dysfunction1-4 but how this relates to cancer progression in humans is unknown. We used a multidisciplinary approach to infuse 13C-labeled nutrients during surgical tumour resection in over 70 patients with kidney cancer. Labeling from [U-13C]glucose varies across cancer subtypes, indicating that the kidney environment alone cannot account for all metabolic reprogramming in these tumours. Compared to the adjacent kidney, clear cell renal cell carcinomas (ccRCC) display suppressed labelling of tricarboxylic acid (TCA) cycle intermediates in vivo and in organotypic slices cultured ex vivo, indicating that suppressed labeling is tissue intrinsic. Infusions of [1,2-13C]acetate and [U-13C]glutamine in patients, coupled with respiratory flux of mitochondria isolated from kidney and tumour tissue, reveal primary defects in mitochondrial function in human ccRCC. However, ccRCC metastases unexpectedly have enhanced labeling of TCA cycle intermediates compared to primary ccRCCs, indicating a divergent metabolic program during ccRCC metastasis in patients. In mice, stimulating respiration in ccRCC cells is sufficient to promote metastatic colonization. Altogether, these findings indicate that metabolic properties evolve during human kidney cancer progression, and suggest that mitochondrial respiration may be limiting for ccRCC metastasis but not for ccRCC growth at the site of origin.

Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway.

Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration.

A male-derived nonribosomal peptide pheromone controls female schistosome development.

Schistosomes cause morbidity and death throughout the developing world due to the massive numbers of eggs female worms deposit into the blood of their host. Studies dating back to the 1920s show that female schistosomes rely on constant physical contact with a male worm both to become and remain sexually mature; however, the molecular details governing this process remain elusive. Here, we uncover a nonribosomal peptide synthetase that is induced in male worms upon pairing with a female and find that it is essential for the ability of male worms to stimulate female development. We demonstrate that this enzyme generates ß-alanyl-tryptamine that is released by paired male worms. Furthermore, synthetic ß-alanyl-tryptamine can replace male worms to stimulate female sexual development and egg laying. These data reveal that peptide-based pheromone signaling controls female schistosome sexual maturation, suggesting avenues for therapeutic intervention and uncovering a role for nonribosomal peptides as metazoan signaling molecules.

Mitochondrial NADP+ is essential for proline biosynthesis during cell growth.

Nicotinamide adenine dinucleotide phosphate (NADP+) is vital to produce NADPH, a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. NADPH exists in separate pools, in both the cytosol and mitochondria; however, the cellular functions of mitochondrial NADPH are incompletely described. Here, we find that decreasing mitochondrial NADP(H) levels through depletion of NAD kinase 2 (NADK2), an enzyme responsible for production of mitochondrial NADP+, renders cells uniquely proline auxotrophic. Cells with NADK2 deletion fail to synthesize proline, due to mitochondrial NADPH deficiency. We uncover the requirement of mitochondrial NADPH and NADK2 activity for the generation of the pyrroline-5-carboxylate metabolite intermediate as the bottleneck step in the proline biosynthesis pathway. Notably, after NADK2 deletion, proline is required to support nucleotide and protein synthesis, making proline essential for the growth and proliferation of NADK2-deficient cells. Thus, we highlight proline auxotrophy in mammalian cells and discover that mitochondrial NADPH is essential to enable proline biosynthesis.

Using arterial-venous analysis to characterize cancer metabolic consumption in patients.

Understanding tumor metabolism holds the promise of new insights into cancer biology, diagnosis and treatment. To assess human cancer metabolism, here we report a method to collect intra-operative samples of blood from an artery directly upstream and a vein directly downstream of a brain tumor, as well as samples from dorsal pedal veins of the same patients. After performing targeted metabolomic analysis, we characterize the metabolites consumed and produced by gliomas in vivo by comparing the arterial supply and venous drainage. N-acetylornithine, D-glucose, putrescine, and L-acetylcarnitine are consumed in relatively large amounts by gliomas. Conversely, L-glutamine, agmatine, and uridine 5-monophosphate are produced in relatively large amounts by gliomas. Further we verify that D-2-hydroxyglutarate (D-2HG) is high in venous plasma from patients with isocitrate dehydrogenases1 (IDH1) mutations. Through these paired comparisons, we can exclude the interpatient variation that is present in plasma samples usually taken from the cubital vein.