Formate-tetrahydrofolate ligase: supplying the cytosolic one-carbon network in roots with one-carbon units originating from glycolate.

The metabolism of tetrahydrofolate (H4PteGlun)-bound one-carbon (C1) units (C1 metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C1 units in specific organelles and tissues. One possible source of C1 units is via formate-tetrahydrofolate ligase, which catalyzes the reversible ATP-driven production of 10-formyltetrahydrofolate (10-formyl-H4PteGlun) from formate and tetrahydrofolate (H4PteGlun). Here, we report biochemical and functional characterization of the enzyme from Arabidopsis thaliana (AtFTHFL). We show that the recombinant AtFTHFL has lower Km and kcat values with pentaglutamyl tetrahydrofolate (H4PteGlu5) as compared to monoglutamyl tetrahydrofolate (H4PteGlu1), resulting in virtually identical catalytic efficiencies for the two substrates. Stable transformation of Arabidopsis plants with the EGFP-tagged AtFTHFL, followed with fluorescence microscopy, demonstrated cytosolic signal. Two independent T-DNA insertion lines with impaired AtFTHFL function had shorter roots compared to the wild type plants, demonstrating the importance of this enzyme for root growth. Overexpressing AtFTHFL led to the accumulation of H4PteGlun + 5,10-methylene-H4PteGlun and serine, accompanied with the depletion of formate and glycolate, in roots of the transgenic Arabidopsis plants. This metabolic adjustment supports the hypothesis that AtFTHFL feeds the cytosolic C1 network in roots with C1 units originating from glycolate, and that these units are then used mainly for biosynthesis of serine, and not as much for the biosynthesis of 5-methyl-H4PteGlun, methionine, and S-adenosylmethionine. This finding has implications for any future attempts to engineer one-carbon unit-requiring products through manipulation of the one-carbon metabolic network in non-photosynthetic organs.

Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Assaying plant formate-tetrahydrofolate ligase with monoglutamylated and polyglutamylated substrates using a fluorescence-HPLC based method.

Formate-tetrahydrofolate ligase catalyzes reversible, ATP-dependent conversion of tetrahydrofolate and formate to 10-formyltetrahydrofolate, simultaneously releasing ADP and inorganic phosphate. This enzyme has traditionally been assayed in the direction of 10-CHO-tetrahydrofolate formation by lowering pH of the reaction post-incubation, thus converting the product of the reaction to 5,10-methenyltetrahydrofolate, which is then quantified spectrophotometrically. To increase sensitivity of the product detection, which is particularly useful when determining the kinetic parameters of the enzyme with polyglutamylated substrates, we have replaced the spectrophotometric detection with HPLC separation and fluorescence detection. In addition to the modified enzyme assay protocol, we are also providing protocols for producing recombinant formate-tetrahydrofolate ligase from Arabidopsis in Escherichia coli cells, producing crude Arabidopsis leaf and root extracts suitable for assaying this enzyme, and for synthesis of polyglutamylated tetrahydrofolate substrates.

Performance of Paracoccus pantotrophus MA3 in heterotrophic nitrification-anaerobic denitrification using formic acid as a carbon source.

Excess amount of nitrogen in wastewater has caused serious concerns, such as water eutrophication. Paracoccus pantotrophus MA3, a novel isolated strain of heterotrophic nitrification-anaerobic denitrification bacteria, was evaluated for nitrogen removal using formic acid as the sole carbon source. The results showed that the maximum ammonium removal efficiency was observed under the optimum conditions of 26.25 carbon to nitrogen ratio, 3.39% (v/v) inoculation amount, 34.64 °C temperature, and at 180 rpm shaking speed, respectively. In addition, quantitative real-time PCR technique analysis assured that the gene expression level of formate dehydrogenase, formate tetrahydrofolate ligase, 5,10-methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, respiratory nitrate reductase beta subunit, L-glutamine synthetase, glutamate dehydrogenase, and glutamate synthase were up-regulated compared to the control group, and combined with nitrogen mass balance analysis to conclude that most of the ammonium was removed by assimilation. A small amount of nitrate and nearly no nitrite were accumulated during heterotrophic nitrification. MA3 exhibited significant denitrification potential under anaerobic conditions with a maximum nitrate removal rate of 4.39 mg/L/h, and the only gas produced was N2. Additionally, 11.50 ± 0.06 mg/L/h of NH4+-N removal rate from biogas slurry was achieved.

Arginine methylation of MTHFD1 by PRMT5 enhances anoikis resistance and cancer metastasis.

Metastasis accounts for the major cause of cancer-related mortality. How disseminated tumor cells survive under suspension conditions and avoid anoikis is largely unknown. Here, using a metabolic enzyme-centered CRISPR-Cas9 genetic screen, we identified methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1) as a novel suppressor of anoikis. MTHFD1 depletion obviously restrained the capacity of cellular antioxidant defense and inhibited tumor distant metastasis. Mechanistically, MTHFD1 was found to bind the protein arginine methyltransferase 5 (PRMT5) and then undergo symmetric dimethylation on R173 by PRMT5. Under suspension conditions, the interaction between MTHFD1 and PRMT5 was strengthened, which increased the symmetric dimethylation of MTHFD1. The elevated methylation of MTHFD1 largely augmented its metabolic activity to generate NADPH, therefore leading to anoikis resistance and distant organ metastasis. Therapeutically, genetic depletion or pharmacological inhibition of PRMT5 declined tumor distant metastasis. And R173 symmetric dimethylation status was associated with metastasis and prognosis of ESCC patients. In conclusion, our study uncovered a novel regulatory role and therapeutic implications of PRMT5/MTHFD1 axis in facilitating anoikis resistance and cancer metastasis.

Serine catabolism generates liver NADPH and supports hepatic lipogenesis.

Carbohydrate can be converted into fat by de novo lipogenesis, a process upregulated in fatty liver disease. Chemically, de novo lipogenesis involves polymerization and reduction of acetyl-CoA, using NADPH as the electron donor. The feedstocks used to generate acetyl-CoA and NADPH in lipogenic tissues remain, however, unclear. Here we show using stable isotope tracing in mice that de novo lipogenesis in adipose is supported by glucose and its catabolism via the pentose phosphate pathway to make NADPH. The liver, in contrast, derives acetyl-CoA for lipogenesis from acetate and lactate, and NADPH from folate-mediated serine catabolism. Such NADPH generation involves the cytosolic serine pathway in liver running in the opposite direction to that observed in most tissues and tumours, with NADPH made by the SHMT1-MTHFD1-ALDH1L1 reaction sequence. SHMT inhibition decreases hepatic lipogenesis. Thus, liver folate metabolism is distinctively wired to support cytosolic NADPH production and lipogenesis. More generally, while the same enzymes are involved in fat synthesis in liver and adipose, different substrates are used, opening the door to tissue-specific pharmacological interventions.

Oncogenic Ras expression increases cellular formate production.

One-carbon units, critical intermediates for cell growth, may be produced by a variety of means, one of which is via the production of formate. Excessive formate accumulation, known as formate overflow and a characteristic of oxidative cancer, has been observed in cancer cells. However, the basis for this high rate of formate production is unknown. We examined the effect of elevated expression of oncogenic Ras (RasV12), on formate production in NIH-3T3 cells (mouse fibroblasts) cultured with either labelled 13C-serine or 13C-glycine. Formate accumulation by the fibroblasts transformed by RasV12 was increased two-threefold over those by vector control (Babe) cells. The production of formate exceeded the rate of utilization in both cell types. 13C-formate was produced almost exclusively from the #3 carbon of 13C-serine. Virtually no labelled formate was produced from either the #2 carbon of serine or the #2 carbon of glycine. The increased formate production by RasV12 cells was associated with increased mRNA abundances for enzymes of formate production in both the mitochondria and the cytosol. Thus, we find the oncogenic RasV12 significantly increases formate overflow and may be one way for tumor cells to produce one-carbon units required for enhanced proliferation of these cells and/or for other processes which have not been identified.

Melatonin modulates metabolic remodeling in HNSCC by suppressing MTHFD1L-formate axis.

Metabolic remodeling is now widely recognized as a hallmark of cancer, yet its role in head and neck squamous cell carcinoma (HNSCC) remains largely unknown. In this study, metabolomic analysis of melatonin-treated HNSCC cell lines revealed that exogenous melatonin inhibited many important metabolic pathways including folate cycle in HNSCC cells. Methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L), a metabolic enzyme of the folate cycle regulating the production of formate, was identified as a downstream target of melatonin. MTHFD1L was found to be markedly upregulated in HNSCC, and MTHFD1L overexpression was significantly associated with unfavorable clinical outcome of HNSCC patients. In addition, MTHFD1L promoted HNSCC progression in vitro and in vivo and reversed the oncostatic effects of exogenous melatonin. More importantly, the malignant phenotypes suppressed by knockdown of MTHFD1L or exogenous melatonin could be partially rescued by formate. Furthermore, we found that melatonin inhibited the expression of MTHFD1L in HNSCC cells through the downregulation of cyclic AMP-responsive element-binding protein 1 (CREB1) phosphorylation. Lastly, this novel regulatory axis of melatonin-p-CREB1-MTHFD1L-formate was also verified in HNSCC tissues. Collectively, our findings have demonstrated that MTHFD1L-formate axis promotes HNSCC progression and melatonin inhibits HNSCC progression through CREB1-mediated downregulation of MTHFD1L and formate. These findings have revealed new metabolic mechanisms in HNSCC and may provide novel insights on the therapeutic intervention of HNSCC.

Association between SNPs and hepatotoxicity in patients with primary central nervous system lymphoma on high-dose methotrexate therapy.

OBJECTIVES: This study aims to evaluate the association between polymorphisms of methotrexate pathway genes and high-dose methotrexate-related hepatotoxicity in Chinese patients with primary central nervous system lymphoma. METHODS: Sixty-five patients in 411 treatment courses were enrolled and their toxicities were evaluated. The association between 30 candidate SNPs from 20 methotrexate pathway genes and high-dose methotrexate-related hepatotoxicity was analysed by PLINK and logistic regression. KEY FINDINGS: TYMS 6 bp DI + II (rs151264360; OR, 0.41; 95% CI, 0.25-0.66; P = 0.00029), MTHFD1 1958 GA + AA (rs2236225; OR, 0.55; 95% CI, 0.33-0.91; P = 0.020) and CCND1 870 GA + GG (rs9344; OR, 0.42; 95% CI, 0.24-0.73; P = 0.0024) had less risk of hepatotoxicity compared with their homozygotes (DD, GG and AA, respectively), while ABCC2 intron 29 GA + GG (rs3740065; OR, 3.14; 95% CI, 1.89-5.20; P = 0.00001) was more prevalent in patients with hepatotoxicity than TT. CONCLUSIONS: TYMS 6 bp DI + II, MTHFD1 1958 GA + AA, CCND1 870 GA + GG genotypes were associated with a lower probability of hepatotoxicity in patients with primary central nervous system lymphoma on high-dose methotrexate therapy, and ABCC2 intron 29 GA + GG was correlated with increased risk of hepatotoxicity.

Triglyceride regulate ACE2 level through MTHFD1.

Obesity has been followed with interest as a risk factor for COVID-19, with triglycerides as one of four common criteria used to define obesity, which have been used to study the mechanism of obesity. In this study, we showed that angiotensin-converting enzyme-2 (ACE2) is widely expressed in the mouse body, including the kidney, spleen, brain, heart, lung, liver, and testis, and that ACE2 levels increased after a high-fat diet. The ACE2 levels were recorded at 0 days, 3 days, 7 days, and 14 days after a high-fat diet, and they increased at 14 days after high-fat diet initiation. In addition, triglyceride levels were also significantly increased at 14 days after high-fat diet initiation, but body weight was not changed. Furthermore, we examined the ACE2 levels in Calu3 cells (a lung cancer cell line) after triglyceride treatment, and the results indicated that ACE2 levels were increased at 25 µM and reached their peak at 200 µM. Finally, we found that the mRNA level of mthfd1 was significantly increased in the high-fat diet group. Given these findings, we hypothesize that triglycerides can regulate the expression of ACE2 and Mthfd1.

Integrated bioinformatics analysis identified MTHFD1L as a potential biomarker and correlated with immune infiltrates in hepatocellular carcinoma.

Liver hepatocellular carcinoma (LIHC) is one of the most frequently occurring primary malignant liver tumors and seriously harms people's health in the world. Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) has been shown to be associated with colon cancer cell proliferation, colony formation and invasion. In the present study, a total of 370 LIHC and 51 normal samples data were downloaded from The Cancer Genome Atlas (TCGA) database. Bioinformatics and immunohistochemistry (IHC) analysis showed that MTHFD1L is highly expressed in liver tumors. Correlation analysis suggested the differences of vital status between high- and low-expression MTHFD1L groups of LIHC. Univariate and multivariate Cox proportional hazards regression were performed to identify the relationship between clinical characteristics and overall survival (OS). In addition, to explore whether MTHFD1L has an effect on the immune infiltration of LIHC. The correlation between MTHFD1L expression and 24 immune cells were analyzed by ImmuneCellAI database. Furthermore, we combined three databases CIBERSORT, TIMER and ImmuneCellAI to do a comprehensive validation and determined that dendritic cells (DCs) resting, macrophage M0 and macrophage M2 closely related to the expression of MTHFD1L. The results showed that MTHFD1L was a potential prognostic biomarker for LIHC, and could help to elucidate that how the immune microenvironment promotes liver cancer development.

Formyl tetrahydrofolate deformylase affects hydrogen peroxide accumulation and leaf senescence by regulating the folate status and redox homeostasis in rice.

It is well established that an abnormal tetrahydrofolate (THF) cycle causes the accumulation of hydrogen peroxide (H2O2) and leaf senescence, however, the molecular mechanism underlying this relationship remains largely unknown. Here, we reported a novel rice tetrahydrofolate cycle mutant, which exhibited H2O2 accumulation and early leaf senescence phenotypes. Map-based cloning revealed that HPA1 encodes a tetrahydrofolate deformylase, and its deficiency led to the accumulation of tetrahydrofolate, 5-formyl tetrahydrofolate and 10-formyl tetrahydrofolate, in contrast, a decrease in 5,10-methenyl-tetrahydrofolate. The expression of tetrahydrofolate cycle-associated genes encoding serine hydroxymethyl transferase, glycine decarboxylase and 5-formyl tetrahydrofolate cycloligase was significantly down-regulated. In addition, the accumulation of H2O2 in hpa1 was not caused by elevated glycolate oxidation. Proteomics and enzyme activity analyses further revealed that mitochondria oxidative phosphorylation complex I and complex V were differentially expressed in hpa1, which was consistent with the H2O2 accumulation in hpa1. In a further feeding assay with exogenous glutathione (GSH), a non-enzymatic antioxidant that consumes H2O2, the H2O2 accumulation and leaf senescence phenotypes of hpa1 were obviously compensated. Taken together, our findings suggest that the accumulation of H2O2 in hpa1 may be mediated by an altered folate status and redox homeostasis, subsequently triggering leaf senescence.

Biochemical properties and crystal structure of formate-tetrahydrofolate ligase from Methylobacterium extorquens CM4.

Methylobacterium extorquens is a methylotroph model organism that has the ability to assimilate formate using the tetrahydrofolate (THF) pathway. The formate-tetrahydrofolate ligase from M. extorquens (MeFtfL) is an enzyme involved in the THF pathway that catalyzes the conversion of formate, THF, and ATP into formyltetrahydrofolate and ADP. To investigate the biochemical properties of MeFtfL, we evaluated the metal usage and enzyme kinetics of the enzyme. MeFtfL uses the Mg ion for catalytic activity, but also has activity for Mn and Ca ions. The enzyme kinetics analysis revealed that Km value of farmate was much higher than THF and ATP, which shows that the ligation activity of MeFtfL is highly dependent on formation concentration. We also determined the crystal structure of MeFtfL at 2.8 Å resolution. MeFtfL functions as a tetramer, and each monomer consists of three domains. The structural superposition of MeFtfL with FtfL from Moorella thermoacetica allowed us to predict the substrate binding site of the enzyme.

MTHFD1L as a folate cycle enzyme correlates with prognostic outcome and its knockdown impairs cell invasive behaviors in osteosarcoma via mediating the AKT/mTOR pathway.

Osteosarcoma (OS) is the most frequent primary malignancy initially in bone with multiple genomic aberrations. Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) is linked with the progression of diverse tumors. However, its function in OS is not understood completely. The expression pattern and prognostic significance of MTHFD1L in OS tissues were analyzed based on GEO database. The expression level of MTHFD1L in OS cell lines was explored by qRT-PCR. The cell proliferation, colony formation ability, invasion as well as migration in OS cells after MTHFD1L knockdown were determined using cell counting kit 8 (CCK-8) assay, colony formation and transwell methods. GSEA analysis was performed to predict the underlying mechanisms of MTHFD1L in OS development. Furthermore, the western blot was utilized to study the influence of MTHFD1L on AKT/mTOR pathway. Our results indicated that MTHFD1L expression was significantly up-regulated in OS tissues and cells compared with normal tissues and cells. High expression of MTHFD1L could lead to poor prognosis of OS patients. Cell proliferation, colony formation ability, migration and invasion were blocked because of reduced MTHFD1L in vitro. Moreover, cell cycle and AKT/mTOR pathway were all associated with MTHFD1L expression. In conclusion, the findings revealed that MTHFD1L might promote the development of OS via mediating cell cycle and AKT/mTOR pathway, indicating that MTHFD1L might act as a promising therapeutic target for OS treatment.

Deletion of neural tube defect-associated gene Mthfd1l causes reduced cranial mesenchyme density.

BACKGROUND: Periconceptional intake of supplemental folic acid can reduce the incidence of neural tube defects by as much as 70%, but the mechanisms by which folic acid supports cellular processes during neural tube closure are unknown. The mitochondrial 10-formyl-tetrahydrofolate synthetase MTHFD1L catalyzes production of formate, thus generating one-carbon units for cytoplasmic processes. Deletion of Mthfd1l causes embryonic lethality, developmental delay, and neural tube defects in mice. METHODS: To investigate the role of mitochondrial one-carbon metabolism during cranial neural tube closure, we have analyzed cellular morphology and function in neural tissues in Mthfd1l knockout embryos. RESULTS: The head mesenchyme showed significantly lower cellular density in Mthfd1l nullizygous embryos compared to wildtype embryos during the process of neural tube closure. Apoptosis and neural crest cell specification were not affected by deletion of Mthfd1l. Sections from the cranial region of Mthfd1l knockout embryos exhibited decreased cellular proliferation, but only after completion of neural tube closure. Supplementation of pregnant dams with formate improved mesenchymal density and corrected cell proliferation in the nullizygous embryos. CONCLUSIONS: Deletion of Mthfd1l causes decreased density in the cranial mesenchyme and this defect is improved with formate supplementation. This study reveals a mechanistic link between folate-dependent mitochondrially produced formate, head mesenchyme formation and neural tube defects.

hnRNPC regulates cancer-specific alternative cleavage and polyadenylation profiles.

Alternative cleavage and polyadenylation (APA) can occur at more than half of all human genes, greatly enhancing the cellular repertoire of mRNA isoforms. As these isoforms can have altered stability, localisation and coding potential, deregulation of APA can disrupt gene expression and this has been linked to many diseases including cancer progression. How APA generates cancer-specific isoform profiles and what their physiological consequences are, however, is largely unclear. Here we use a subcellular fractionation approach to determine the nuclear and cytoplasmic APA profiles of successive stages of colon cancer using a cell line-based model. Using this approach, we show that during cancer progression specific APA profiles are established. We identify that overexpression of hnRNPC has a critical role in the establishment of APA profiles characteristic for metastatic colon cancer cells, by regulating poly(A) site selection in a subset of genes that have been implicated in cancer progression including MTHFD1L.

Rewiring of one carbon metabolism in Salmonella serves as an excellent live vaccine against systemic salmonellosis.

Live attenuated vaccines are superior to the killed or subunit vaccines. We designed a Salmonella Typhimurium strain by deleting folD gene (encoding methylenetetrahydrofolate dehydrogenase-cyclohydrolase) in the presence of a heterologous fhs gene (encoding formyltetrahydrofolate synthetase) and tested its vaccine potential under stringent conditions of lethal and sub-lethal challenges with virulent Salmonella in the murine model. The efficacy of the vaccine in conferring protection against Salmonella infection was determined in a wide range of host conditions of systemic infection, corresponding to human young adults, neonates, geriatric age and, importantly, to the immune compromised state of pregnancy. The standardized vaccination regime comprised a primary dose of 104 CFU/animal followed by a booster dose of 102 CFU/animal on day 7. Challenge with the virulent pathogen was done at day 7 post-administration of the booster. Subsequently, the mortality, morbidity, systemic colonization, antibody response and cytokine profiling were determined. The vaccinated cohort showed a strong protection against virulent pathogen in all models tested. The serum anti-Salmonella antibody titers and cytokine levels were significantly higher in the vaccinated cohort compared to the mock vaccinated cohort. Thus, we report the development and validation of a live attenuated vaccine candidate conferring excellent protection against Salmonellosis and typhoid fever.

Assimilation of formic acid and CO2 by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways.

Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO2 can be an attractive raw material for bio-based chemicals. Here, we report the development of Escherichia coli strains assimilating FA and CO2 through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The Methylobacterium extorquens formate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA assimilation. The gcv reaction was reversed by knocking out the repressor gene (gcvR) and overexpressing the gcvTHP genes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO2 in a glucose-containing medium. Native serine deaminase converted serine to pyruvate, showing 4.5% of pyruvate-forming flux comes from FA and CO2 The pyruvate-forming flux from FA and CO2 could be increased to 14.9% by knocking out gcvR, pflB, and serA, chromosomally expressing gcvTHP under trc, and overexpressing the reconstructed THF cycle, gcvTHP, and lpd genes in one vector. To reduce glucose usage required for energy and redox generation, the Candida boidinii formate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO2 consumption rates of 370.2, 145.6, and 14.9 mg⋅g dry cell weight (DCW)-1⋅h-1, respectively. The C1 assimilation pathway consumed 21.3 wt% of FA. Furthermore, cells sustained slight growth using only FA and CO2 after glucose depletion, suggesting that combined use of the C1 assimilation pathway and C. boidinii Fdh will be useful for eventually developing a strain capable of utilizing FA and CO2 without an additional carbon source such as glucose.

EWS-FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine-glycine biosynthesis.

Ewing sarcoma (EWS) is a soft tissue and bone tumor that occurs primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS-FLI1 is the primary oncogenic driver. The epigenome of EWS cells reflects EWS-FLI1 binding and activation or repression of transcription. Here, we demonstrate that EWS-FLI1 positively regulates the expression of proteins required for serine-glycine biosynthesis and uptake of the alternative nutrient source glutamine. Specifically, we show that EWS-FLI1 activates expression of PHGDH, PSAT1, PSPH, and SHMT2. Using cell-based studies, we also establish that EWS cells are dependent on glutamine for cell survival and that EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and two enzymes involved in the one-carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine-glycine biosynthesis in EWS cells impacts their redox state leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned genes we identified as regulated by EWS-FLI1 exhibit increased expression compared with normal tissues. Furthermore, retrospective analysis of an independent data set generated a significant stratification of the overall survival of EWS patients into low- and high-risk groups based on the expression of PHGDH, PSAT1, PSPH, SHMT2, SLC1A5, MTHFD2, and MTHFD1L. In summary, our study demonstrates that EWS-FLI1 reprograms the metabolism of EWS cells and that serine-glycine metabolism or glutamine uptake are potential targetable vulnerabilities in this tumor type.

Low Dietary Folate Interacts with MTHFD1 Synthetase Deficiency in Mice, a Model for the R653Q Variant, to Increase Incidence of Developmental Delays and Defects.

Background: Suboptimal folate intake, a risk factor for birth defects, is common even in areas with folate fortification. A polymorphism in methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), R653Q (MTHFD1 c.1958 G > A), has also been associated with increased birth defect risk, likely through reduced purine synthesis. Objective: We aimed to determine if the interaction of MTHFD1 synthetase deficiency and low folate intake increases developmental abnormalities in a mouse model for MTHFD1 R653Q. Methods: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed control or low-folate diets (2 and 0.3 mg folic acid/kg diet, respectively) before mating and during pregnancy. Embryos and placentas were examined for anomalies at embryonic day 10.5. Maternal 1-carbon metabolites were measured in plasma and liver. Results: Delays and defects doubled in litters of Mthfd1S+/- females fed low-folate diets compared to wild-type females fed either diet, or Mthfd1S+/- females fed control diets [P values (defects): diet 0.003, maternal genotype 0.012, diet × maternal genotype 0.014]. These adverse outcomes were associated with placental dysmorphology. Intrauterine growth restriction was increased by embryonic Mthfd1S+/- genotype, folate deficiency, and interaction of maternal Mthfd1S+/- genotype with folate deficiency (P values: embryonic genotype 0.045, diet 0.0081, diet × maternal genotype 0.0019). Despite a 50% increase in methylenetetrahydrofolate reductase expression in low-folate maternal liver (P diet = 0.0007), methyltetrahydrofolate concentration decreased 70% (P diet <0.0001) and homocysteine concentration doubled in plasma (P diet = 0.0001); S-adenosylmethionine decreased 40% and S-adenosylhomocysteine increased 20% in low-folate maternal liver (P diet = 0.002 and 0.0002, respectively). Conclusions: MTHFD1 synthetase-deficient mice are more sensitive to low folate intake than wild-type mice during pregnancy. Reduced purine synthesis due to synthetase deficiency and altered methylation potential due to low folate may increase pregnancy complications. Further studies and individualized intake recommendations may be required for women homozygous for the MTHFD1 R653Q variant.