FAMetA: a mass isotopologue-based tool for the comprehensive analysis of fatty acid metabolism.

The use of stable isotope tracers and mass spectrometry (MS) is the gold standard method for the analysis of fatty acid (FA) metabolism. Yet, current state-of-the-art tools provide limited and difficult-to-interpret information about FA biosynthetic routes. Here we present FAMetA, an R package and a web-based application (www.fameta.es) that uses 13C mass isotopologue profiles to estimate FA import, de novo lipogenesis, elongation and desaturation in a user-friendly platform. The FAMetA workflow covers the required functionalities needed for MS data analyses. To illustrate its utility, different in vitro and in vivo experimental settings are used in which FA metabolism is modified. Thanks to the comprehensive characterization of FA biosynthesis and the easy-to-interpret graphical representations compared to previous tools, FAMetA discloses unnoticed insights into how cells reprogram their FA metabolism and, when combined with FASN, SCD1 and FADS2 inhibitors, it enables the identification of new FAs by the metabolic reconstruction of their synthesis route.

MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

CAR T-Cells Depend on the Coupling of NADH Oxidation with ATP Production.

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell's expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.

Tumor Microenvironment-Derived Metabolites: A Guide to Find New Metabolic Therapeutic Targets and Biomarkers.

Metabolic reprogramming is a hallmark of cancer that enables cancer cells to grow, proliferate and survive. This metabolic rewiring is intrinsically regulated by mutations in oncogenes and tumor suppressors, but also extrinsically by tumor microenvironment factors (nutrient and oxygen availability, cell-to-cell interactions, cytokines, hormones, etc.). Intriguingly, only a few cancers are driven by mutations in metabolic genes, which lead metabolites with oncogenic properties (i.e., oncometabolites) to accumulate. In the last decade, there has been rekindled interest in understanding how dysregulated metabolism and its crosstalk with various cell types in the tumor microenvironment not only sustains biosynthesis and energy production for cancer cells, but also contributes to immune escape. An assessment of dysregulated intratumor metabolism has long since been exploited for cancer diagnosis, monitoring and therapy, as exemplified by 18F-2-deoxyglucose positron emission tomography imaging. However, the efficient delivery of precision medicine demands less invasive, cheaper and faster technologies to precisely predict and monitor therapy response. The metabolomic analysis of tumor and/or microenvironment-derived metabolites in readily accessible biological samples is likely to play an important role in this sense. Here, we review altered cancer metabolism and its crosstalk with the tumor microenvironment to focus on energy and biomass sources, oncometabolites and the production of immunosuppressive metabolites. We provide an overview of current pharmacological approaches targeting such dysregulated metabolic landscapes and noninvasive approaches to characterize cancer metabolism for diagnosis, therapy and efficacy assessment.

SHMT inhibition is effective and synergizes with methotrexate in T-cell acute lymphoblastic leukemia.

Folate metabolism enables cell growth by providing one-carbon (1C) units for nucleotide biosynthesis. The 1C units are carried by tetrahydrofolate, whose production by the enzyme dihydrofolate reductase is targeted by the important anticancer drug methotrexate. 1C units come largely from serine catabolism by the enzyme serine hydroxymethyltransferase (SHMT), whose mitochondrial isoform is strongly upregulated in cancer. Here we report the SHMT inhibitor SHIN2 and demonstrate its in vivo target engagement with 13C-serine tracing. As methotrexate is standard treatment for T-cell acute lymphoblastic leukemia (T-ALL), we explored the utility of SHIN2 in this disease. SHIN2 increases survival in NOTCH1-driven mouse primary T-ALL in vivo. Low dose methotrexate sensitizes Molt4 human T-ALL cells to SHIN2, and cells rendered methotrexate resistant in vitro show enhanced sensitivity to SHIN2. Finally, SHIN2 and methotrexate synergize in mouse primary T-ALL and in a human patient-derived xenograft in vivo, increasing survival. Thus, SHMT inhibition offers a complementary strategy in the treatment of T-ALL.

Obesity Shapes Metabolism in the Tumor Microenvironment to Suppress Anti-Tumor Immunity.

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.

Chaperone-mediated autophagy regulates the pluripotency of embryonic stem cells.

Embryonic stem cells can propagate indefinitely in a pluripotent state, able to differentiate into all types of specialized cells when restored to the embryo. What sustains their pluripotency during propagation remains unclear. Here, we show that core pluripotency factors OCT4 and SOX2 suppress chaperone-mediated autophagy (CMA), a selective form of autophagy, until the initiation of differentiation. Low CMA activity promotes embryonic stem cell self-renewal, whereas its up-regulation enhances differentiation. CMA degrades isocitrate dehydrogenases IDH1 and IDH2 and reduces levels of intracellular α-ketoglutarate, an obligatory cofactor for various histone and DNA demethylases involved in pluripotency. These findings suggest that CMA mediates the effect of core pluripotency factors on metabolism, shaping the epigenetic landscape of stem cells and governing the balance between self-renewal and differentiation.

A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway.

Glucose is catabolized by two fundamental pathways, glycolysis to make ATP and the oxidative pentose phosphate pathway to make reduced nicotinamide adenine dinucleotide phosphate (NADPH). The first step of the oxidative pentose phosphate pathway is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here we develop metabolite reporter and deuterium tracer assays to monitor cellular G6PD activity. Using these, we show that the most widely cited G6PD antagonist, dehydroepiandosterone, does not robustly inhibit G6PD in cells. We then identify a small molecule (G6PDi-1) that more effectively inhibits G6PD. Across a range of cultured cells, G6PDi-1 depletes NADPH most strongly in lymphocytes. In T cells but not macrophages, G6PDi-1 markedly decreases inflammatory cytokine production. In neutrophils, it suppresses respiratory burst. Thus, we provide a cell-active small molecule tool for oxidative pentose phosphate pathway inhibition, and use it to identify G6PD as a pharmacological target for modulating immune response.

Lactate dehydrogenase inhibition synergizes with IL-21 to promote CD8+ T cell stemness and antitumor immunity.

Interleukin (IL)-2 and IL-21 dichotomously shape CD8+ T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (TSCM) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1 While deletion of Ldha prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of TSCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21.

The Tumor Metabolic Microenvironment: Lessons from Lactate.

The extracellular milieu of tumors is generally assumed to be immunosuppressive due in part to metabolic factors. Here, we review methods for probing the tumor metabolic microenvironment. In parallel, we consider the resulting available evidence, with a focus on lactate, which is the most strongly increased metabolite in bulk tumors. Limited microenvironment concentration measurements suggest depletion of glucose and modest accumulation of lactate (less than 2-fold). Isotope tracer measurements show rapid lactate exchange between the tumor and circulation. Such exchange is catalyzed by MCT transporters, which cotransport lactate and protons (H+). Rapid lactate exchange seems at odds with tumor lactate accumulation. We propose a potential resolution to this paradox. Because of the high pH of tumor cells relative to the microenvironment, H+-coupled transport by MCTs tends to drive lactate from the interstitium into tumor cells. Accordingly, lactate may accumulate preferentially in tumor cells, not the microenvironment. Thus, although they are likely subject to other immunosuppressive metabolic factors, tumor immune cells may not experience a high lactate environment. The lack of clarity regarding microenvironmental lactate highlights the general need for careful metabolite measurements in the tumor extracellular milieu.

PPARγ is a nexus controlling alternative activation of macrophages via glutamine metabolism.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is known to regulate lipid metabolism in many tissues, including macrophages. Here we report that peritoneal macrophage respiration is enhanced by rosiglitazone, an activating PPARγ ligand, in a PPARγ-dependent manner. Moreover, PPARγ is required for macrophage respiration even in the absence of exogenous ligand. Unexpectedly, the absence of PPARγ dramatically affects the oxidation of glutamine. Both glutamine and PPARγ have been implicated in alternative activation (AA) of macrophages, and PPARγ was required for interleukin 4 (IL4)-dependent gene expression and stimulation of macrophage respiration. Indeed, unstimulated macrophages lacking PPARγ contained elevated levels of the inflammation-associated metabolite itaconate and express a proinflammatory transcriptome that, remarkably, phenocopied that of macrophages depleted of glutamine. Thus, PPARγ functions as a checkpoint, guarding against inflammation, and is permissive for AA by facilitating glutamine metabolism. However, PPARγ expression is itself markedly increased by IL4. This suggests that PPARγ functions at the center of a feed-forward loop that is central to AA of macrophages.

Metabolomics discloses donor liver biomarkers associated with early allograft dysfunction.

BACKGROUND & AIMS: Early allograft dysfunction (EAD) dramatically influences graft and patient outcome after orthotopic liver transplantation and its incidence is strongly determined by donor liver quality. Nevertheless, objective biomarkers, which can assess graft quality and anticipate organ function, are still lacking. This study aims to investigate whether there is a preoperative donor liver metabolomic biosignature associated with EAD. METHODS: A comprehensive metabolomic profiling of 124 donor liver biopsies collected before transplantation was performed by mass spectrometry coupled to liquid chromatography. Donor liver grafts were classified into two groups: showing EAD and immediate graft function (IGF). Multivariate data analysis was used to search for the relationship between the metabolomic profiles present in donor livers before transplantation and their function in recipients. RESULTS: A set of liver graft dysfunction-associated biomarkers was identified. Key changes include significantly increased levels of bile acids, lysophospholipids, phospholipids, sphingomyelins and histidine metabolism products, all suggestive of disrupted lipid homeostasis and altered histidine pathway. Based on these biomarkers, a predictive EAD model was built and further evaluated by assessing 24 independent donor livers, yielding 91% sensitivity and 82% specificity. The model was also successfully challenged by evaluating donor livers showing primary non-function (n=4). CONCLUSIONS: A metabolomic biosignature that accurately differentiates donor livers, which later showed EAD or IGF, has been deciphered. The remarkable metabolomic differences between donor livers before transplant can relate to their different quality. The proposed metabolomic approach may become a clinical tool for donor liver quality assessment and for anticipating graft function before transplant.

Mammalian cell metabolomics: experimental design and sample preparation.

Metabolomics represents the global assessment of metabolites in a biological sample and reports the closest information to the phenotype of the biological system under study. Mammalian cell metabolomics has emerged as a promising tool with potential applications in many biotechnology and research areas. Metabolomics workflow includes experimental design, sampling, sample processing, metabolite analysis, and data processing. Given their influence on metabolite content and biological interpretation of data, a good experimental design and the appropriate choice of a sample processing method are prerequisites for success in any metabolomic study. The use of mammalian cells in the metabolomics field involves harder sample processing methods, including metabolism quenching and metabolite extraction, as compared to the use of body fluids, although such critical issues are frequently overlooked. This review aims to overview the common experimental procedures used in mammalian cell metabolomics based on mass spectrometry, by placing special emphasis on discussing sample preparation approaches, although other aspects, such as cell metabolomics applications, culture systems, cellular models, analytical platforms, and data analysis, are also briefly covered. This review intends to be a helpful tool to assist researchers in addressing decisions when planning a metabolomics study involving the use of mammalian cells.

Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method.

Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA profiles in different species (human, rat, and mouse) by quantifying 31 major and minor BA species in a single 21-min run. The method has been validated according to FDA guidelines, and it generally provides good results in terms of intra- and interday precision (less than 8.6% and 16.0%, respectively), accuracy (relative error measurement between -11.9% and 8.6%), and linearity (R(2) > 0.996 and dynamic ranges between two and four orders of magnitude), with limits of quantification between 2.5 and 20 nM. The new analytical approach was applied to determine BA concentrations in human, rat, and mouse serum and in liver tissue. Our comparative study confirmed and extended previous reports, showing marked interspecies differences in circulating and hepatic BA composition. The targeted analysis revealed the presence of unexpected minoritary BAs, such as tauro-alpha-Muricholic acid in human serum, thus allowing us to obtain a thorough profiling of human samples. Its great sensitivity, low sample requirements (25 µl of serum, 5 mg of tissue), and comprehensive capacity to profile a considerable number of BAs make the present method a good choice to study BA metabolism in physiological and pathological situations, particularly in toxicological studies.

A comprehensive untargeted metabonomic analysis of human steatotic liver tissue by RP and HILIC chromatography coupled to mass spectrometry reveals important metabolic alterations.

Steatosis, or excessive accumulation of lipids in the liver, is a generally accepted previous step to the development of more severe conditions like nonalcoholic steatohepatitis, fibrosis, and cirrhosis. We aimed to characterize the metabolic profile that defines simple steatosis in human tissue and to identify potential disturbances in the hepatic metabolism that could favor the switch to progressive liver damage. A total of 46 samples, 23 from steatotic and 23 from nonsteatotic human livers, were analyzed following a holistic LC-MS-based metabonomic analysis that combines RP and HILIC chromatographic separations. Multivariate statistical data analysis satisfactorily classified samples and revealed steatosis-associated biomarkers. Increased levels of bile acids and phospholipid degradation products, and decreased levels of antioxidant species, were found in steatotic livers, indicating disturbances in lipid and bile acid homeostasis and mitochondrial dysfunction. Changes in hypoxanthine, creatinine, glutamate, glutamine, or γ-glutamyl-dipeptides concentrations, suggestive of alterations in energy metabolism and amino acid metabolism and transport, were also found. The results show that the proposed analytical strategy is suitable to achieve a comprehensive metabolic profile of steatotic human liver tissue and provide new insights into the metabolic alterations occurring in fatty liver that could contribute to its predisposition to damage evolution.