A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

Crosstalk between mechanotransduction and metabolism.

Mechanical forces shape cells and tissues during development and adult homeostasis. In addition, they also signal to cells via mechanotransduction pathways to control cell proliferation, differentiation and death. These processes require metabolism of nutrients for both energy generation and biosynthesis of macromolecules. However, how cellular mechanics and metabolism are connected is still poorly understood. Here, we discuss recent evidence indicating how the mechanical cues exerted by the extracellular matrix (ECM), cell-ECM and cell-cell adhesion complexes influence metabolic pathways. Moreover, we explore the energy and metabolic requirements associated with cell mechanics and ECM remodelling, implicating a reciprocal crosstalk between cell mechanics and metabolism.

Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages.

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.

Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes.

Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.

Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

The context-specific roles of urea cycle enzymes in tumorigenesis.

The expression of the urea cycle (UC) proteins is dysregulated in multiple cancers, providing metabolic benefits to tumor survival, proliferation, and growth. Here, we review the main changes described in the expression of UC enzymes and metabolites in different cancers at various stages and suggest that these changes are dynamic and should hence be viewed in a context-specific manner. Understanding the evolvability in the activity of the UC pathway in cancer has implications for cancer-immune cell interactions and for cancer diagnosis and therapy.

Functional implications of fumarate-induced cysteine succination.

Mutations in metabolic enzymes are associated with hereditary and sporadic forms of cancer. For example, loss-of-function mutations affecting fumarate hydratase (FH), the tricarboxylic acid (TCA) cycle enzyme, result in the accumulation of millimolar levels of fumarate that cause an aggressive form of kidney cancer. A distinct feature of fumarate is its ability to spontaneously react with thiol groups of cysteines in a chemical reaction termed succination. Although succination of a few proteins has been causally implicated in the molecular features of FH-deficient cancers, the stoichiometry, wider functional consequences, and contribution of succination to disease development remain largely unexplored. We discuss the functional implications of fumarate-induced succination in FH-deficient cells, the available methodologies, and the current challenges in studying this post-translational modification.

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency.

Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity.

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.

Convergent somatic mutations in metabolism genes in chronic liver disease.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

Hypoxia promotes osteogenesis by facilitating acetyl-CoA-mediated mitochondrial-nuclear communication.

Bone-derived mesenchymal stem cells (MSCs) reside in a hypoxic niche that maintains their differentiation potential. While hypoxia (low oxygen concentration) was reported to critically support stem cell function and osteogenesis, the molecular events triggering changes in stem cell fate decisions in response to normoxia (high oxygen concentration) remain elusive. Here, we study the impact of normoxia on mitochondrial-nuclear communication during stem cell differentiation. We show that normoxia-cultured murine MSCs undergo profound transcriptional alterations which cause irreversible osteogenesis defects. Mechanistically, high oxygen promotes chromatin compaction and histone hypo-acetylation, particularly on promoters and enhancers of osteogenic genes. Although normoxia induces metabolic rewiring resulting in elevated acetyl-CoA levels, histone hypo-acetylation occurs due to the trapping of acetyl-CoA inside mitochondria owing to decreased citrate carrier (CiC) activity. Restoring the cytosolic acetyl-CoA pool remodels the chromatin landscape and rescues the osteogenic defects. Collectively, our results demonstrate that the metabolism-chromatin-osteogenesis axis is perturbed upon exposure to high oxygen levels and identifies CiC as a novel, oxygen-sensitive regulator of the MSC function.

NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction.

The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Signaling metabolite L-2-hydroxyglutarate activates the transcription factor HIF-1α in lipopolysaccharide-activated macrophages.

Activated macrophages undergo metabolic reprogramming, which not only supports their energetic demands but also allows for the production of specific metabolites that function as signaling molecules. Several Krebs cycles, or Krebs-cycle-derived metabolites, including succinate, α-ketoglutarate, and itaconate, have recently been shown to modulate macrophage function. The accumulation of 2-hydroxyglutarate (2HG) has also been well documented in transformed cells and more recently shown to play a role in T cell and dendritic cell function. Here we have found that the abundance of both enantiomers of 2HG is increased in LPS-activated macrophages. We show that L-2HG, but not D-2HG, can promote the expression of the proinflammatory cytokine IL-1ß and the adoption of an inflammatory, highly glycolytic metabolic state. These changes are likely mediated through activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) by L-2HG, a known inhibitor of the HIF prolyl hydroxylases. Expression of the enzyme responsible for L-2HG degradation, L-2HG dehydrogenase (L-2HGDH), was also found to be decreased in LPS-stimulated macrophages and may therefore also contribute to L-2HG accumulation. Finally, overexpression of L-2HGDH in HEK293 TLR4/MD2/CD14 cells inhibited HIF-1α activation by LPS, while knockdown of L-2HGDH in macrophages boosted the induction of HIF-1α-dependent genes, as well as increasing LPS-induced HIF-1α activity. Taken together, this study therefore identifies L-2HG as a metabolite that can regulate HIF-1α in macrophages.

Fumarate hydratase (FH) and cancer: a paradigm of oncometabolism.

Fumarate hydratase (FH) is an enzyme of the Tricarboxylic Acid (TCA) cycle whose mutations lead to hereditary and sporadic forms of cancer. Although more than twenty years have passed since its discovery as the leading cause of the cancer syndrome Hereditary leiomyomatosis and Renal Cell Carcinoma (HLRCC), it is still unclear how the loss of FH causes cancer in a tissue-specific manner and with such aggressive behaviour. It has been shown that FH loss, via the accumulation of FH substrate fumarate, activates a series of oncogenic cascades whose contribution to transformation is still under investigation. In this review, we will summarise these recent findings in an integrated fashion and put forward the case that understanding the biology of FH and how its mutations promote transformation will be vital to establish novel paradigms of oncometabolism.