1C Pathway Research Articles

Tracing the Diverse Paths of One-Carbon Metabolism in Cancer and Beyond.

One-carbon (1C) metabolism is a network of biochemical reactions distributed across organelles that delivers folate-activated 1C units to support macromolecule synthesis, methylation, and reductive homeostasis. Fluxes through these pathways are up-regulated in highly proliferative cancer cells, and anti-folates, which target enzymes within the 1C pathway, have long been used in the treatment of cancer. In this work, we review fundamental aspects of 1C metabolism and place it in context with other biosynthetic and redox pathways, such that 1C metabolism acts to bridge pathways across compartments. We further discuss the importance of stable-isotope-tracing techniques combined with mass spectrometry analysis to study 1C metabolism and conclude by highlighting therapeutic approaches that could exploit cancer cells' dependency on 1C metabolism.

Microbe-derived Antioxidants Enhance Lipid Synthesis by Regulating the Hepatic AMPKα–SREBP1c Pathway in Weanling Piglets

BackgroundWeaning usually causes low feed intake and weight loss in piglets, which mobilizes lipid to energize. The microbe-derived antioxidants (MAs) exhibit great potential in antioxidation, anti-inflammation, and metabolic regulation. ObjectivesWe aimed to investigate the changes of lipid metabolism postweaning and effects of MA on growth performance and hepatic lipid metabolism in weanling piglets. MethodsIn the first experiment, piglets weaned at 21 d of age were slaughtered on weaning day (d0), 4 (d4), and 14 (d14) postweaning (6 piglets per day). In the second experiment, piglets were divided into 2 groups, receiving MA (MA) and saline gavage (CON), respectively. All piglets were weaned at 21 d of age and 6 piglets from each group were slaughtered at 25 d of age. ResultsIn experiment 1, the serum triglyceride, total cholesterol (TC), and LDL cholesterol on d4 and d14 declined significantly compared with d0 (P < 0.05). The serum leptin on d0 was higher than that on d4 and d14 (P < 0.05). The serum ghrelin kept increasing from d0 to d14 (P < 0.05). The hepatic hormone-sensitive lipase and adipose triglyceride lipase first increased from d0 to d4 and then decreased from d4 to d14 (P < 0.05). In experiment 2, the average daily gain and average daily feed intake from 21 to 25 d of age increased in the MA group compared with the CON group (P < 0.05). The serum TC, hepatic TC, and glucose of MA group showed a significant increase than that of the CON group (P < 0.05). The expression of SCD1, ACAT2, and PPARγ were upregulated in the MA group (P < 0.05). Contrary to the decreased expression of phosphorylation of adenosine 5′-monophosphate-activated protein kinase alfa subunit (Thr172), the nuclear sterol regulatory element–binding protein 1c, fatty acid synthase, and peroxisome proliferator-activated receptor gamma of MA group increased than that of CON group (P < 0.05). ConclusionsWeaning promoted hepatic lipolysis and MA could enhance lipid synthesis by regulating adenosine 5′-monophosphate-activated protein kinase alfa subunit–sterol regulatory element–binding protein 1c pathway, thus improving growth performance of weanling piglets.

Overexpression of salusin‑α upregulates AdipoR2 and activates the PPARα/ApoA5/SREBP‑1c pathway to inhibit lipid synthesis in HepG2 cells.

Salusin‑α and adiponectin, are vasoactive peptides with numerous similar biological effects related to lipid metabolism. Adiponectin has been shown to reduce fatty acid oxidation and to inhibit lipid synthesis of liver cells through its receptor, adiponectin receptor2 (AdipoR2), but whether salusin‑α is able to interact with AdipoR2, was not previously reported. To investigate this, invitro experiments were carried out. The overexpression and interference recombinant plasmids were constructed with salusin‑α. The lentiviral expression systems of salusin‑α overexpression and interference were respectively synthesized in 293Tcells, and 293Tcells were infected with the lentivirus. Finally, the association between salusin‑α and AdipoR2 was analyzed by semi‑quantitative PCR. Subsequently, HepG2 cells were also infected with these viruses. The expression levels of AdipoR2, peroxisome proliferator‑activated receptor‑α (PPARα), apolipoproteinA5 (ApoA5) and sterol regulatory element‑binding transcription factor1 (SREBP‑1c) were detected by western blotting, and AdipoR2 inhibitor (thapsigargin) and agonist [4‑phenyl butyric acid (PBA)] were used to observe the resultant changes in the aforementioned molecules. The results obtained revealed that the overexpression of salusin‑α increased the level of AdipoR2 in 293TandHepG2cells, led to an upregulation of the levels of PPARα and ApoA5, and inhibited the expression of SREBP‑1c, whereas the salusin‑α interference lentivirus exerted the opposite effects. Notably, thapsigargin inhibited the expression of AdipoR2, PPARα and ApoA5 in HepG2 cells of pHAGE‑Salusin‑α group, and caused an increase in the level of SREBP‑1c, whereas the opposite effects were observed in pLKO.1‑shSalusin‑α#1 group upon treatment with PBA. Taken together, these data demonstrated that overexpression of salusin‑α upregulated AdipoR2, which in turn activated the PPARα/ApoA5/SREBP‑1c signaling pathway to inhibit lipid synthesis in HepG2 cells, thereby providing theoretical data on which to base the clinical application of salusin‑α as a novel peptide for molecular intervention in fatty liver disease.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Downregulation of phosphoserine phosphatase potentiates tumor immune environments to enhance immune checkpoint blockade therapy

BackgroundEffects of immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) are limited. The current study explored the possibility of exploiting tumor metabolic switches to enhance HCC sensitivity to immune...

TMET-35. ONE CARBON AND NUCLEOTIDE METABOLISM ARE NOVEL TARGETS IN PEDIATRIC BRAINSTEM TUMORS

Abstract Diffuse intrinsic pontine glioma (DIPG) are inoperable tumors of the brainstem with no cure. The median survival of children with DIPG is less than 2 years and 5-year overall survival is only 1%. Genomic studies have identified recurrent mutations in histone H3, ACVR and TP53 genes in DIPG. However, there are still no breakthrough drug targets for DIPG. To better understand the disease biology and find new drug targets for DIPG, we recently performed joint pathway analysis of DIPG metabolites (untargeted metabolomics) and differentially expressed genes (RNAseq) relative to normal human neural stem cells. We discovered that specific enzymes of the de novo purine biosynthesis pathway (DNPB), serine synthesis and the associated and one-carbon (1C) metabolism are highly active and the most upregulated in DIPG tumors and cell lines. Gain and loss of function experiments as well as pharmacological studies indicated a mechanistic link between DIPG mutations and DNPB pathway and the dependence of DIPG cells on specific DNPB and 1C metabolism enzymes for in vitro and in vivo growth. The 1C pathway is crucial for rapidly proliferating malignant cells as it senses and regulates cellular nutrient status by allocating and cycling 1C-groups between different acceptor compounds. It controls synthesis of nucleotides, amino acids, glycogen and phosphoglyceride precursors, S-adenosylmethionine (SAM), glutathione and NADPH. Thus, besides dispensing carbon atoms it also regulates cellular epigenetic and redox status. An important carbon donor is serine which is synthesized from glucose at 3-phosphoglycerate step in glycolysis. Untargeted as well as carbon and nitrogen tracing experiments indicated large folate pools in DIPG cells that can serve as a buffer to potentially blunt antifolate therapy. Experiments are underway to understand the importance of the folate cycle in DIPG and design novel and translatable combination therapies for DIPG.

Tamoxifen induced hepatic steatosis in high-fat feeding rats through SIRT1-Foxo1 suppression and LXR-SREBP1c activation.

Clinically, long-term use of tamoxifen (TAM) would lead to fatty liver disease in breast cancer patients, especially obese women. However, the exact mechanism of TAM-induced hepatic steatosis is still unclear. Meanwhile, there is no drug to prevent and treat it. In view of silent information regulator 1 (SIRT1) playing a key role in hepatic lipid metabolism regulation, this study was conducted to investigate whether SIRT1 is a potential therapeutic target for TAM-induced hepatic steatosis. In this study, obese female Wistar rats fed with high-fat diet (HFD) for 15weeks were given TAM (4, 8mg/kg, intragastric) for 14days. In vitro, human hepatocarcinoma cell line HepG2 was used to establish a high-fat model with 50μM oleic acid and TAM (10μM) was treated simultaneously for 72h. The results showed that TAM was more likely to upregulate the expression of lipid synthetase that caused the increase of lipid content in HepG2 cells and rat liver. The expression of SIRT1 was downregulated both in vitro and in vivo. SIRT1 agonist SRT1720 (15mg/kg, 30mg/kg, i.p.) could resist TAM-induced hepatic lipid synthetase overexpression to relieve TAM-induced hepatic steatosis. Meanwhile, the upregulation of p-forkhead box O1 and LXRα induced by TAM was reversed by SRT1720. These results indicated that TAM-induced hepatic steatosis was based on SIRT1-p-FoxO/LXRα-sterol regulatory element binding protein 1c pathway under HFD condition. SIRT1 agonist might be a potential therapeutic drug to relieve this side effect. Tamoxifen increased lipid synthesis and regulated lipid transport in HFD rat liver.p-FoxO1/LXRα-SREBP1c signaling was upregulated through the inhibition of SIRT1 in tamoxifen-induced hepatic steatosis under HFD condition.SIRT1 agonist SRT1720 could relieve tamoxifen-induced hepatic steatosis.

Myristica fragrans Extract Regulates Gut Microbes and Metabolites to Attenuate Hepatic Inflammation and Lipid Metabolism Disorders via the AhR-FAS and NF-κB Signaling Pathways in Mice with Non-Alcoholic Fatty Liver Disease.

Recent studies have shown that non-alcoholic fatty liver disease (NAFLD) is closely related to the gut microbiome. Myristica fragrans is widely used as a traditional seasoning and has a therapeutic effect on gastrointestinal diseases. Although previous studies have shown that M. fragrans extracts have anti-obesity and anti-diabetes effects in mice fed a high-fat diet, few studies have determined the active components or the corresponding mechanism in vivo. In this study, for the first time, an M. fragrans extract (MFE) was shown to be a prebiotic that regulates gut microbes and metabolites in mice fed a high-fat diet. Bioinformatics, network pharmacology, microbiome, and metabolomics analyses were used to analyze the nutrient–target pathway interactions in mice with NAFLD. The National Center for Biotechnology Information Gene Expression Omnibus database was used to analyze NAFLD-related clinical data sets to predict potential targets. The drug database and disease database were then integrated to perform microbiome and metabolomics analyses to predict the target pathways. The concentrations of inflammatory factors in the serum and liver, such as interleukin-6 and tumor necrosis factor-α, were downregulated by MFE. We also found that the hepatic concentrations of low-density lipoprotein cholesterol, total cholesterol, and triglycerides were decreased after MFE treatment. Inhibition of the nuclear factor kappa B (NF-κB) pathway and downregulation of the fatty acid synthase (FAS)-sterol regulatory element-binding protein 1c pathway resulted in the regulation of inflammation and lipid metabolism by activating tryptophan metabolite–mediated aryl hydrocarbon receptors (AhR). In summary, MFE effectively attenuated inflammation and lipid metabolism disorders in mice with NAFLD through the NF-κB and AhR–FAS pathways.

Ancestry-Specific Interactions Between Circulatory Folate and One-Carbon Metabolism Genes’ Haplotypes for Higher-Grade Cervical Intraepithelial Neoplasia

Abstract Objectives Determine ancestry-specific interactions between circulatory folate concentrations, haplotypes of the one-carbon (1C) pathway genes and risk of higher-grade cervical intraepithelial neoplasia (CIN2+) in the US post-folic acid fortification era. Methods Study included self-reported African American and Caucasian American women positive for high-risk human papillomavirus (HPV) genotypes and diagnosed with ≤ CIN1 (non-cases, n = 340) or CIN2+ (cases, n = 337). Plasma and red blood cell (RBC) folate, vitamins B12 (B12) and C, and total carotene levels were measured. 660 single nucleotide polymorphisms of the 1C pathway genes and 104 ancestry informative markers (AIMs) were analyzed using buffy coat DNA and customizable Illumina GoldenGate arrays. Global African ancestry (GA) was estimated using the AIMs. Ancestry-based African American (AFR) had GA ≥ 0.8 and European Americans (EA) otherwise. Common haplotype blocks (n = 50) were tested for interactions with circulatory folate using logistic regression adjusting for age, education, body mass index (BMI), body fat %, smoking, parity, hormonal contraception use, plasma total carotene, B12 and C. False discovery rate &amp;lt; 0.1 was considered significant. Results Some of the main findings were, increasing plasma folate (1 SD) decreases the risk of CIN2 + when homozygous for haplotype CG at (rs575425, rs586199) of BHMT gene in EA (odds ratio/OR = 0.13, CI = 0.04,0.44), and for haplotype TA at (rs559062, rs515064) of CTH in AFR (OR = 0.23, CI = 0.1,0.52); increasing RBC folate (1 SD) decreases risk of CIN2 + when homozygous for haplotypes TA at (rs559062, rs515064) of CTH (OR = 0.27, CI = 0.12,0.61), AC at (rs7706298, rs10512934) of MTRR (OR = 0.21, CI = 0.08,0.57), and AGA at (rs11672909, rs759920, rs7253062) of DNMT1 (OR = 0.4, CI = 0.18,0.90) in AFR; and increasing RBC folate (1 SD) increases the risk of CIN2 + when homozygous for haplotypes GG at (rs3856027, rs4650051) of CTH gene (OR = 3.52, CI = 1.27,9.71), and CGG at (rs11672909, rs759920, rs7253062) of DNMT1 (OR = 5.70, CI = 1.86,17.49) in AFR. Conclusions Circulatory folate differentially modulates the risk for CIN2 + by interacting with haplotypes of the 1C pathway genes according to ancestry in reproductive-aged women exposed to US folic acid fortification program. Funding Sources National Cancer Institute

Dietary obesity in mice is associated with lipid deposition and metabolic shifts in the lungs sharing features with the liver

Obesity is associated with both chronic and acute respiratory illnesses, such as asthma, chronic obstructive pulmonary disease (COPD) or increased susceptibility to infectious diseases. Anatomical but also systemic and local metabolic alterations are proposed contributors to the pathophysiology of lung diseases in the context of obesity. To bring perspective to this discussion, we used NMR to compare the obesity-associated metabolomic profiles of the lung with those of the liver, heart, skeletal muscles, kidneys, brain and serum from male C57Bl/6J mice fed with a high-fat and high-sucrose (HFHSD) diet vs. standard (SD) chow for 14 weeks. Our results showed that the lung was the second most affected organ after the liver, and that the two organs shared reduced one-carbon (1C) metabolism and increased lipid accumulation. Altered 1C metabolism was found in all organs and in the serum, but serine levels were increased only in the lung of HFHSD compared to SD. Lastly, tricarboxylic acid (TCA)-derived metabolites were specifically and oppositely regulated in the serum and kidneys but not in other organs. Collectively, our data highlighted that HFHSD induced specific metabolic changes in all organs, the lung being the second most affected organ, the main alterations affecting metabolite concentrations of the 1C pathway and, to a minor extend, TCA. The absolute metabolite quantification performed in this study reveals some metabolic specificities affecting both the liver and the lung, that may reveal common metabolic determinants to the ongoing pathological process.

Therapeutic targeting of the mitochondrial one-carbon pathway: perspectives, pitfalls, and potential.

Most of the drugs currently prescribed for cancer treatment are riddled with substantial side effects. In order to develop more effective and specific strategies to treat cancer, it is of importance to understand the biology of drug targets, particularly the newly emerging ones. A comprehensive evaluation of these targets will benefit drug development with increased likelihood for success in clinical trials. The folate-mediated one-carbon (1C) metabolism pathway has drawn renewed attention as it is often hyperactivated in cancer and inhibition of this pathway displays promise in developing anticancer treatment with fewer side effects. Here, we systematically review individual enzymes in the 1C pathway and their compartmentalization to mitochondria and cytosol. Based on these insight, we conclude that (1) except the known 1C targets (DHFR, GART, and TYMS), MTHFD2 emerges as good drug target, especially for treating hematopoietic cancers such as CLL, AML, and T-cell lymphoma; (2) SHMT2 and MTHFD1L are potential drug targets; and (3) MTHFD2L and ALDH1L2 should not be considered as drug targets. We highlight MTHFD2 as an excellent therapeutic target and SHMT2 as a complementary target based on structural/biochemical considerations and up-to-date inhibitor development, which underscores the perspectives of their therapeutic potential.

Interspecific Variation in One-Carbon Metabolism within the Ovarian Follicle, Oocyte, and Preimplantation Embryo: Consequences for Epigenetic Programming of DNA Methylation.

One-carbon (1C) metabolism provides methyl groups for the synthesis and/or methylation of purines and pyrimidines, biogenic amines, proteins, and phospholipids. Our understanding of how 1C pathways operate, however, pertains mostly to the (rat) liver. Here we report that transcripts for all bar two genes (i.e., BHMT, MAT1A) encoding enzymes in the linked methionine-folate cycles are expressed in all cell types within the ovarian follicle, oocyte, and blastocyst in the cow, sheep, and pig; as well as in rat granulosa cells (GCs) and human KGN cells (a granulosa-like tumor cell line). Betaine-homocysteine methyltransferase (BHMT) protein was absent in bovine theca and GCs, as was activity of this enzyme in GCs. Mathematical modeling predicted that absence of this enzyme would lead to more volatile S-adenosylmethionine-mediated transmethylation in response to 1C substrate (e.g., methionine) or cofactor provision. We tested the sensitivity of bovine GCs to reduced methionine (from 50 to 10 µM) and observed a diminished flux of 1C units through the methionine cycle. We then used reduced-representation bisulfite sequencing to demonstrate that this reduction in methionine during bovine embryo culture leads to genome-wide alterations to DNA methylation in >1600 genes, including a cohort of imprinted genes linked to an abnormal fetal-overgrowth phenotype. Bovine ovarian and embryonic cells are acutely sensitive to methionine, but further experimentation is required to determine the significance of interspecific variation in BHMT expression.

Therapeutic Targeting of Mitochondrial One-Carbon Metabolism in Cancer.

One-carbon (1C) metabolism encompasses folate-mediated 1C transfer reactions and related processes, including nucleotide and amino acid biosynthesis, antioxidant regeneration, and epigenetic regulation. 1C pathways are compartmentalized in the cytosol, mitochondria, and nucleus. 1C metabolism in the cytosol has been an important therapeutic target for cancer since the inception of modern chemotherapy, and "antifolates" targeting cytosolic 1C pathways continue to be a mainstay of the chemotherapy armamentarium for cancer. Recent insights into the complexities of 1C metabolism in cancer cells, including the critical role of the mitochondrial 1C pathway as a source of 1C units, glycine, reducing equivalents, and ATP, have spurred the discovery of novel compounds that target these reactions, with particular focus on 5,10-methylene tetrahydrofolate dehydrogenase 2 and serine hydroxymethyltransferase 2. In this review, we discuss key aspects of 1C metabolism, with emphasis on the importance of mitochondrial 1C metabolism to metabolic homeostasis, its relationship with the oncogenic phenotype, and its therapeutic potential for cancer.

Translational control of one-carbon metabolism underpins ribosomal protein phenotypes in cell division and longevity.

A long-standing problem is how cells that lack one of the highly similar ribosomal proteins (RPs) often display distinct phenotypes. Yeast and other organisms live longer when they lack specific ribosomal proteins, especially of the large 60S subunit of the ribosome. However, longevity is neither associated with the generation time of RP deletion mutants nor with bulk inhibition of protein synthesis. Here, we queried actively dividing RP mutants through the cell cycle. Our data link transcriptional, translational, and metabolic changes to phenotypes associated with the loss of paralogous RPs. We uncovered translational control of transcripts encoding enzymes of methionine and serine metabolism, which are part of one-carbon (1C) pathways. Cells lacking Rpl22Ap, which are long-lived, have lower levels of metabolites associated with 1C metabolism. Loss of 1C enzymes increased the longevity of wild type cells. 1C pathways exist in all organisms and targeting the relevant enzymes could represent longevity interventions.

Alisol A attenuates high-fat-diet-induced obesity and metabolic disorders via the AMPK/ACC/SREBP-1c pathway.

Obesity and its associated metabolic disorders such as diabetes, hepatic steatosis and chronic heart diseases are affecting billions of individuals. However there is no satisfactory drug to treat such diseases. In this study, we found that alisol A, a major active triterpene isolated from the Chinese traditional medicine Rhizoma Alismatis, could significantly attenuate high‐fat‐diet‐induced obesity. Our biochemical detection demonstrated that alisol A remarkably decreased lipid levels, alleviated glucose metabolism disorders and insulin resistance in high‐fat‐diet‐induced obese mice. We also found that alisol A reduced hepatic steatosis and improved liver function in the obese mice model.In addition, protein expression investigation revealed that alisol A had an active effect on AMPK/ACC/SREBP‐1c pathway. As suggested by the molecular docking study, such bioactivity of alisol A may result from its selective binding to the catalytic region of AMPK.Therefore, we believe that Alisol A could serve as a promising agent for treatment of obesity and its related metabolic diseases.

Powering Epigenetics through the 1C Pathway.

Epigenetic modifications in plants repress transposable elements to maintain genome stability and facilitate adaptation to changing environmental conditions by regulating the expression of some genes. Furthermore, conditions such as disease stress can alter the epigenetic machinery to impact gene

Deletion of the neural tube defect–associated gene Mthfd1l disrupts one-carbon and central energy metabolism in mouse embryos

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion-induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism.

Fatty acids promote fatty liver disease via the dysregulation of 3-mercaptopyruvate sulfurtransferase/hydrogen sulfide pathway

ObjectiveAccumulation of free fatty acids (FFAs) in hepatocytes induces lipotoxicity, leading to non-alcoholic fatty liver disease (NAFLD). This study aimed to investigate the underlying mechanisms by which FFA contributes to...

Abstract 232: Pharmacological and genetic dissection of one carbon pathway provides novel insights into generation, transformation and utilization of one carbon units

Abstract Tumorigenesis, tumor progression and metastasis require metabolic rewiring to support the need for rapid biomass accumulation, reduction of oxidative stress and epigenetic remodeling. One carbon (1C) metabolism provides essential substrates for such metabolic rewiring and enzymes of 1C pathway are frequently overexpressed in tumors consistent with the increased demands on these biochemical pathways. While 1C pathway modulators such as antifolates are efficacious, the clinical utility of antifolates is limited by their inability to distinguish between the metabolic requirements of proliferating cells vs transformed cancer cells. Precise targeting of 1C metabolism in transformed cells can potentially lead to highly effective and safe anticancer therapies. The enzymes of 1C metabolism are organized in a spatially distinct set of parallel reactions in the cytosol and the mitochondrion. The core 1C machinery extracts 1C units from donors such as serine onto a folate backbone. The oxidation state of the folate associated 1C unit can then be manipulated according to the needs of the 1C utilization reactions. The quantitative contribution of cytosolic vs mitochondrial pathway, regulation of 1C metabolism and biochemical basis of partitioning of 1C units between different downstream reactions is not fully understood. To understand the role of the mitochondrial and cytosolic pathways in the generation and transformation of 1C units, we generated potent and selective inhibitors of MethyleneTetraHydroFolate Dehydrogenase Type 2 (MTHFD2) and dual inhibitors of Serine HydroxyMethyl Transferase (SHMT) 1 and 2. We also generated knockout cells lacking various enzymes of mitochondrial and cytosolic 1C pathway. The combined pharmacological-genetic approach allowed quantitative dissection of the contribution of cytosolic and mitochondrial pathways. Interestingly, genetic disruption of the mitochondrial pathway dramatically increased sensitivity of cells to inhibition of SHMT1 while deletion of SHMT1 increased the sensitivity to MTHFD2 inhibitors suggesting interdependence and flexibility between the mitochondrial and cytosolic 1C pathway arms. The effects of 1C inhibitors on cell viability could be rescued by other 1C donors as well as 1C pathway products, further informing on the critical products of 1C metabolism and potential escape mechanisms. Our observations show that cancer cells can utilize multiple routes to generate 1C units and identify the contribution of specific enzymes. Pharmacological inhibition of some of these enzymes strongly affects cell viability and combination of inhibitors of cytosolic and mitochondrial 1C enzymes in the same or different molecules can be particularly effective. In addition, genetic lesions in the mitochondrial or cytosolic 1C arms can also sensitize tumors to inhibitors of specific 1C pathway enzymes. Citation Format: Vipin Suri, Nello Mainolfi, Adam Friedman, Mikel P. Moyer, Greg Ducker, Joshua D. Rabinowitz, Mark Manfredi. Pharmacological and genetic dissection of one carbon pathway provides novel insights into generation, transformation and utilization of one carbon units. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 232.

Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway

One-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here, we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-tetrahydrofolate (10-formyl-THF), and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis, with the mitochondrial pathway required in nutrient-poor conditions.