Reductive carboxylation supports growth in tumour cells with defective mitochondria

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucoseand glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP/ NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetylcoenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containingmutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells withmutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacologicalETC inhibition.Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism. Wefirst studied themetabolismof isogenic143Bhumanosteosarcoma cells that contained or lacked a loss-of-functionmutation in ETC complex III (cytochrome b–c1 complex). These cell lines were generated by depleting 143B mitochondrial DNA (mtDNA) and repopulating with either wild-type mtDNA or mtDNA containing a frameshift mutation in the gene encoding cytochrome b (CYTB: also known asMT-CYB), an essential complex III component. Despite lack of respiration and complex III function in themutants, bothwild-type (WT143B) andCYTBmutant (CYTB 143B) cells form colonies in soft agar and proliferate at comparable rates (Supplementary Fig. 1a), making these cells a good model in which to study growth during mitochondrial dysfunction. Both cell lines had detectable CAC intermediates, although citrate was less abundant and succinate was significantly more abundant in the CYTB 143B cells (Supplementary Fig. 1b). As expected for cells with defective oxidative phosphorylation, CYTB 143B cells had higher glucose consumption and lactate production thanWT 143B cells, indicating a metabolic shift towards aerobic glycolysis (Fig. 1a). To determine the effects ofCYTBmutation on themetabolic fates of glucose, we cultured both cell lines inmedium containing D[U-C]glucose (hereU indicates uniformly labelled) and measured C enrichment of intracellular metabolites by mass spectrometry. In WT 143B cells, most citrate molecules contained glucose-derived C (Fig. 1b). Citrate m12 (citrate containing two additional mass units from C) results from oxidative decarboxylation of glucose-derived pyruvate by pyruvate dehydrogenase (PDH) to form [1,2-C]acetyl-coA (coA, coenzyme A), followed by condensation with an unlabelled