Integrating molecular and clinical variables to predict myocardial recovery.

Mechanical unloading and circulatory support with left ventricular assist devices (LVADs) mediate significant myocardial improvement in a subset of advanced heart failure (HF) patients. The clinical and biological phenomena associated with cardiac recovery are under intensive investigation. Left ventricular (LV) apical tissue, alongside clinical data, were collected from HF patients at the time of LVAD implantation (n=208). RNA was isolated and mRNA transcripts were identified through RNA sequencing and confirmed with RT-qPCR. To our knowledge this is the first study to combine transcriptomic and clinical data to derive predictors of myocardial recovery. We used a bioinformatic approach to integrate 59 clinical variables and 22,373 mRNA transcripts at the time of LVAD implantation for the prediction of post-LVAD myocardial recovery defined as LV ejection fraction (LVEF) ≥40% and LV end-diastolic diameter (LVEDD) ≤5.9cm, as well as functional and structural LV improvement independently by using LVEF and LVEDD as continuous variables, respectively. To substantiate the predicted variables, we used a multi-model approach with logistic and linear regressions. Combining RNA and clinical data resulted in a gradient boosted model with 80 features achieving an AUC of 0.731±0.15 for predicting myocardial recovery. Variables associated with myocardial recovery from a clinical standpoint included HF duration, pre-LVAD LVEF, LVEDD, and HF pharmacologic therapy, and LRRN4CL (ligand binding and programmed cell death) from a biological standpoint. Our findings could have diagnostic, prognostic, and therapeutic implications for advanced HF patients, and inform the care of the broader HF population.

Enhancing mitochondrial pyruvate metabolism ameliorates myocardial ischemic reperfusion injury.

The established clinical therapy for the treatment of acute myocardial infarction is primary percutaneous coronary intervention (PPCI) to restore blood flow to the ischemic myocardium. PPCI is effective at reperfusing the ischemic myocardium, however the rapid re-introduction of oxygenated blood also can cause ischemia-reperfusion (I/R) injury. Reperfusion injury is the culprit for up to half of the final myocardial damage, but there are no clinical interventions to reduce I/R injury. We previously demonstrated that inhibiting the lactate exporter, monocarboxylate transporter 4 (MCT4), and re-directing pyruvate towards oxidation can blunt isoproterenol-induced hypertrophy. Based on this finding, we hypothesized that the same pathway might be important during I/R. Here, we establish that the pyruvate-lactate metabolic axis plays a critical role in determining myocardial salvage following injury. Post-I/R injury, the mitochondrial pyruvate carrier (MPC), required for pyruvate oxidation, is upregulated in the surviving myocardium following I/R injury. MPC loss in cardiomyocytes caused more cell death with less myocardial salvage, which was associated with an upregulation of MCT4 in the myocardium at risk of injury. We deployed a pharmacological strategy of MCT4 inhibition with a highly selective compound (VB124) at the time of reperfusion. This strategy normalized reactive oxygen species (ROS), mitochondrial membrane potential (Δψ), and Ca 2+ , increased pyruvate entry to TCA cycle, and improved myocardial salvage and functional outcomes following I/R injury. Altogether, our data suggest that normalizing the pyruvate-lactate metabolic axis via MCT4 inhibition is a promising pharmacological strategy to mitigate I/R injury.

Glycine homeostasis requires reverse SHMT flux.

The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Using rodent models, here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable-isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered, but SHMT2- and serine-dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis is largely insensitive to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 a major glycine-consuming enzyme.

Phospholipid isotope tracing reveals ß-catenin-driven suppression of phosphatidylcholine metabolism in hepatocellular carcinoma.

Background and Aims: Activating mutations in the CTNNB1 gene encoding ß-catenin are among the most frequently observed oncogenic alterations in hepatocellular carcinoma (HCC). HCC with CTNNB1 mutations show profound alterations in lipid metabolism including increases in fatty acid oxidation and transformation of the phospholipidome, but it is unclear how these changes arise and whether they contribute to the oncogenic program in HCC. Methods: We employed untargeted lipidomics and targeted isotope tracing to quantify phospholipid production fluxes in an inducible human liver cell line expressing mutant ß-catenin, as well as in transgenic zebrafish with activated ß-catenin-driven HCC. Results: In both models, activated ß-catenin expression was associated with large changes in the lipidome including conserved increases in acylcarnitines and ceramides and decreases in triglycerides. Lipid flux analysis in human cells revealed a large reduction in phosphatidylcholine (PC) production rates as assayed by choline tracer incorporation. We developed isotope tracing lipid flux analysis for zebrafish and observed similar reductions in phosphatidylcholine synthesis flux accomplished by sex-specific mechanisms. Conclusions: The integration of isotope tracing with lipid abundances highlights specific lipid class transformations downstream of ß-catenin signaling in HCC and suggests future HCC-specific lipid metabolic targets.

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.

The pyruvate-lactate axis modulates cardiac hypertrophy and heart failure.

The metabolic rewiring of cardiomyocytes is a widely accepted hallmark of heart failure (HF). These metabolic changes include a decrease in mitochondrial pyruvate oxidation and an increased export of lactate. We identify the mitochondrial pyruvate carrier (MPC) and the cellular lactate exporter monocarboxylate transporter 4 (MCT4) as pivotal nodes in this metabolic axis. We observed that cardiac assist device-induced myocardial recovery in chronic HF patients was coincident with increased myocardial expression of the MPC. Moreover, the genetic ablation of the MPC in cultured cardiomyocytes and in adult murine hearts was sufficient to induce hypertrophy and HF. Conversely, MPC overexpression attenuated drug-induced hypertrophy in a cell-autonomous manner. We also introduced a novel, highly potent MCT4 inhibitor that mitigated hypertrophy in cultured cardiomyocytes and in mice. Together, we find that alteration of the pyruvate-lactate axis is a fundamental and early feature of cardiac hypertrophy and failure.

Mitochondrial pyruvate carrier is required for optimal brown fat thermogenesis.

Brown adipose tissue (BAT) is composed of thermogenic cells that convert chemical energy into heat to maintain a constant body temperature and counteract metabolic disease. The metabolic adaptations required for thermogenesis are not fully understood. Here, we explore how steady state levels of metabolic intermediates are altered in brown adipose tissue in response to cold exposure. Transcriptome and metabolome analysis revealed changes in pathways involved in amino acid, glucose, and TCA cycle metabolism. Using isotopic labeling experiments, we found that activated brown adipocytes increased labeling of pyruvate and TCA cycle intermediates from U13C-glucose. Although glucose oxidation has been implicated as being essential for thermogenesis, its requirement for efficient thermogenesis has not been directly tested. We show that mitochondrial pyruvate uptake is essential for optimal thermogenesis, as conditional deletion of Mpc1 in brown adipocytes leads to impaired cold adaptation. Isotopic labeling experiments using U13C-glucose showed that loss of MPC1 led to impaired labeling of TCA cycle intermediates. Loss of MPC1 in BAT increased 3-hydroxybutyrate levels in blood and BAT in response to the cold, suggesting that ketogenesis provides an alternative fuel source to compensate. Collectively, these studies highlight that complete glucose oxidation is essential for optimal brown fat thermogenesis.

Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism.

Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on the breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared with the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared with primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma and kidney chromophobe cell carcinoma, also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit metabolism plays an important role in promoting cancer progression, particularly in late-stage cancer. IMPLICATIONS: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers.

Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier.

Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.

Novel Pyrrolo[3,2-d]pyrimidine Compounds Target Mitochondrial and Cytosolic One-carbon Metabolism with Broad-spectrum Antitumor Efficacy.

Folate-dependent one-carbon (C1) metabolism is compartmentalized into the mitochondria and cytosol and supports cell growth through nucleotide and amino acid biosynthesis. Mitochondrial C1 metabolism, including serine hydroxymethyltransferase (SHMT) 2, provides glycine, NAD(P)H, ATP, and C1 units for cytosolic biosynthetic reactions, and is implicated in the oncogenic phenotype across a wide range of cancers. Whereas multitargeted inhibitors of cytosolic C1 metabolism, such as pemetrexed, are used clinically, there are currently no anticancer drugs that specifically target mitochondrial C1 metabolism. We used molecular modeling to design novel small-molecule pyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1 metabolism at SHMT2. In vitro antitumor efficacy was established with the lead compounds (AGF291, AGF320, AGF347) toward lung, colon, and pancreatic cancer cells. Intracellular targets were identified by metabolic rescue with glycine and nucleosides, and by targeted metabolomics using a stable isotope tracer, with confirmation by in vitro assays with purified enzymes. In addition to targeting SHMT2, inhibition of the cytosolic purine biosynthetic enzymes, ß-glycinamide ribonucleotide formyltransferase and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and SHMT1 was also established. AGF347 generated significant in vivo antitumor efficacy with potential for complete responses against both early-stage and upstage MIA PaCa-2 pancreatic tumor xenografts, providing compelling proof-of-concept for therapeutic targeting of SHMT2 and cytosolic C1 enzymes by this series. Our results establish structure-activity relationships and identify exciting new drug prototypes for further development as multitargeted antitumor agents.

Serine Metabolism Supports Macrophage IL-1ß Production.

Serine is a substrate for nucleotide, NADPH, and glutathione (GSH) synthesis. Previous studies in cancer cells and lymphocytes have shown that serine-dependent one-carbon units are necessary for nucleotide production to support proliferation. Presently, it is unknown whether serine metabolism impacts the function of non-proliferative cells, such as inflammatory macrophages. We find that in macrophages, serine is required for optimal lipopolysaccharide (LPS) induction of IL-1ß mRNA expression, but not inflammasome activation. The mechanism involves a requirement for glycine, which is made from serine, to support macrophage GSH synthesis. Cell-permeable GSH, but not the one-carbon donor formate, rescues IL-1ß mRNA expression. Pharmacological inhibition of de novo serine synthesis in vivo decreased LPS induction of IL-1ß levels and improved survival in an LPS-driven model of sepsis in mice. Our study reveals that serine metabolism is necessary for GSH synthesis to support IL-1ß cytokine production.

A Patient-derived Xenograft Model of Pancreatic Neuroendocrine Tumors Identifies Sapanisertib as a Possible New Treatment for Everolimus-resistant Tumors.

Patients with pancreatic neuroendocrine tumors (PNET) commonly develop advanced disease and require systemic therapy. However, treatment options remain limited, in part, because experimental models that reliably emulate PNET disease are lacking. We therefore developed a patient-derived xenograft model of PNET (PDX-PNET), which we then used to evaluate two mTOR inhibitor drugs: FDA-approved everolimus and the investigational new drug sapanisertib. PDX-PNETs maintained a PNET morphology and PNET-specific gene expression signature with serial passage. PDX-PNETs also harbored mutations in genes previously associated with PNETs (such as MEN1 and PTEN), displayed activation of the mTOR pathway, and could be detected by Gallium-68 DOTATATE PET-CT. Treatment of PDX-PNETs with either everolimus or sapanisertib strongly inhibited growth. As seen in patients, some PDX-PNETs developed resistance to everolimus. However, sapanisertib, a more potent inhibitor of the mTOR pathway, caused tumor shrinkage in most everolimus-resistant tumors. Our PDX-PNET model is the first available, validated PDX model for PNET, and preclinical data from the use of this model suggest that sapanisertib may be an effective new treatment option for patients with PNET or everolimus-resistant PNET.

5,10-methenyltetrahydrofolate synthetase deficiency causes a neurometabolic disorder associated with microcephaly, epilepsy, and cerebral hypomyelination.

Folate metabolism in the brain is critically important and serves a number of vital roles in nucleotide synthesis, single carbon metabolism/methylation, amino acid metabolism, and mitochondrial translation. Genetic defects in almost every enzyme of folate metabolism have been reported to date, and most have neurological sequelae. We report 2 patients presenting with a neurometabolic disorder associated with biallelic variants in the MTHFS gene, encoding 5,10-methenyltetrahydrofolate synthetase. Both patients presented with microcephaly, short stature, severe global developmental delay, progressive spasticity, epilepsy, and cerebral hypomyelination. Baseline CSF 5-methyltetrahydrolate (5-MTHF) levels were in the low-normal range. The first patient was treated with folinic acid, which resulted in worsening cerebral folate deficiency. Treatment in this patient with a combination of oral L-5-methyltetrahydrofolate and intramuscular methylcobalamin was able to increase CSF 5-MTHF levels, was well tolerated over a 4 month period, and resulted in subjective mild improvements in functioning. Measurement of MTHFS enzyme activity in fibroblasts confirmed reduced activity. The direct substrate of the MTHFS reaction, 5-formyl-THF, was elevated 30-fold in patient fibroblasts compared to control, supporting the hypothesis that the pathophysiology of this disorder is a manifestation of toxicity from this metabolite.

Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.

Mitochondrial translation requires folate-dependent tRNA methylation.

Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation.

Human SHMT inhibitors reveal defective glycine import as a targetable metabolic vulnerability of diffuse large B-cell lymphoma.

The enzyme serine hydroxymethyltransferse (SHMT) converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Folate one-carbon units support purine and thymidine synthesis, and thus cell growth. Mammals have both cytosolic SHMT1 and mitochondrial SHMT2, with the mitochondrial isozyme strongly up-regulated in cancer. Here we show genetically that dual SHMT1/2 knockout blocks HCT-116 colon cancer tumor xenograft formation. Building from a pyrazolopyran scaffold that inhibits plant SHMT, we identify small-molecule dual inhibitors of human SHMT1/2 (biochemical IC50 ∼ 10 nM). Metabolomics and isotope tracer studies demonstrate effective cellular target engagement. A cancer cell-line screen revealed that B-cell lines are particularly sensitive to SHMT inhibition. The one-carbon donor formate generally rescues cells from SHMT inhibition, but paradoxically increases the inhibitor's cytotoxicity in diffuse large B-cell lymphoma (DLBCL). We show that this effect is rooted in defective glycine uptake in DLBCL cell lines, rendering them uniquely dependent upon SHMT enzymatic activity to meet glycine demand. Thus, defective glycine import is a targetable metabolic deficiency of DLBCL.

An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate.

The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.

One-Carbon Metabolism in Health and Disease.

One-carbon (1C) metabolism, mediated by the folate cofactor, supports multiple physiological processes. These include biosynthesis (purines and thymidine), amino acid homeostasis (glycine, serine, and methionine), epigenetic maintenance, and redox defense. Both within eukaryotic cells and across organs, 1C metabolic reactions are compartmentalized. Here we review the fundamentals of mammalian 1C metabolism, including the pathways active in different compartments, cell types, and biological states. Emphasis is given to recent discoveries enabled by modern genetics, analytical chemistry, and isotope tracing. An emerging theme is the biological importance of mitochondrial 1C reactions, both for producing 1C units that are exported to the cytosol and for making additional products, including glycine and NADPH. Increased clarity regarding differential folate pathway usage in cancer, stem cells, development, and adult physiology is reviewed and highlights new opportunities for selective therapeutic intervention.