Synthesis Of S-adenosylmethionine Research Articles

Engineering of Methionine Adenosyltransferase toward Mitigated Product Inhibition for Efficient Production of S-Adenosylmethionine.

S-Adenosylmethionine (SAM) is a crucial metabolic intermediate playing irreplaceable roles in organismal activities. However, the synthesis of SAM by methionine adenosyltransferase (MAT) is hindered by low conversion due to severe product inhibition. Herein structure-guided semirational engineering was conducted on MAT from Escherichia coli (EcMAT) to mitigate the product inhibitory effect. Compared with the wild-type EcMAT, the best variant E56Q/Q105R exhibited an 8.13-fold increase in half maximal inhibitory concentration and a 4.46-fold increase in conversion (150 mM ATP and l-methionine), leading to a SAM titer of 47.02 g/L. Another variant, E56N/Q105R, showed superior thermostability with an impressive 85.30-fold increase in half-life (50 °C) value. Furthermore, molecular dynamics (MD) simulation results demonstrate that the alleviation in product inhibitory effect could be attributed to facilitated product release. This study offers molecular insights into the mitigated product inhibition, and provides valuable guidance for engineering MAT toward enhanced catalytic performance.

Betaine alleviates spermatogenic cells apoptosis of oligoasthenozoospermia rat model by up-regulating methyltransferases and affecting DNA methylation

BackgroundOligoasthenozoospermia is the most common type of semen abnormality in male infertile patients. Betaine (BET) has been proved to have pharmacological effects on improving semen quality. BET also belongs to endogenous physiological active substances in the testis. However, the physiological function of BET in rat testis and its pharmacological mechanism against oligoasthenozoospermia remain unclear. PurposeThis research aims to prove the therapeutic effect and potential mechanism of BET on oligoasthenozoospermia rat model induced by Tripterygium wilfordii glycosides (TWGs). MethodsThe oligoasthenozoospermia rat model was established by a continuous gavage of TWGs (60 mg/kg) for 28 days. Negative control group, oligoasthenozoospermia group, positive drug group (levocarnitine, 300 mg/kg), and 200 mg/kg, 400 mg/kg, and 800 mg/kg BET groups were created for exploring the therapeutic effect of BET on the oligoasthenozoospermia rat model. The therapeutic effect was evaluated by HE and TUNEL staining. Immunofluorescence assay of DNMT3A, PIWIL1, PRMT5, SETDB1, BHMT2, and METTL3, methylation capture sequencing, Pi-RNA sequencing, and molecular docking were used to elucidate potential pharmacological mechanisms. ResultsIt is proved that BET can significantly restore testicular pathological damage induced by TWGs, which also can significantly reverse the apoptosis of spermatogenic cells. The spermatogenic cell protein expression levels of DNMT3A, PIWIL1, PRMT5, SETDB1, BHMT2, and METTL3 significantly decreased in oligoasthenozoospermia group. 400 mg/kg and 800 mg/kg BET groups can significantly increase expression level of the above-mentioned proteins. Methylation capture sequencing showed that BET can significantly increase the 5mC methylation level of Spata, Spag, and Specc spermatogenesis-related genes. Pi-RNA sequencing proved that the above-mentioned genes produce a large number of Pi-RNA under BET intervention. Pi-RNA can form complexes with PIWI proteins to participate in DNA methylation of target genes. Molecular docking indicated that BET may not directly act as substrate for methyltransferase and instead participates in DNA methylation by promoting the methionine cycle and increasing S-adenosylmethionine synthesis. ConclusionBET has a significant therapeutic effect on oligoasthenozoospermia rat model induced by TWPs. The mechanism mainly involves that BET can increase the methylation level of Spata, Specc, and Spag target genes through the PIWI/Pi-RNA pathway and up-regulation of methyltransferases (including DNA methyltransferases and histone methyltransferases).

The combination of methionine adenosyltransferase 2A inhibitor and methyltransferase like 3 inhibitor promotes apoptosis of non-small cell lung cancer cells and produces synergistic anti-tumor activity

Methionine adenosyltransferase 2 A (MAT2A) mediates the synthesis of methyl donor S-Adenosylmethionine (SAM), providing raw materials for methylation reactions in cells. MAT2A inhibitors are currently used for the treatment of tumors with methylthioadenosine phosphorylase (MTAP) deficiency in clinical research. Methyltransferase like 3 (METTL3) catalyzes N6-methyladenosine (m6A) modification of mRNA in mammalian cells using SAM as the substrate which has been shown to affect the tumorigenesis of non-small cell lung cancer (NSCLC) from multiple perspectives. MAT2A-induced SAM depletion may have the potential to inhibit the methyl transfer function of METTL3. Therefore, in order to expand the applicability of inhibitors, improve anti-tumor effects and reduce toxicity, the combinational effect of MAT2A inhibitor AG-270 and METTL3 inhibitor STM2457 was evaluated in NSCLC. The results showed that this combination induced cell apoptosis rather than cell cycle arrest, which was non-tissue-specific and was independent of MTAP expression status, resulting in a significant synergistic anti-tumor effect. We further elucidated that the combination-induced enhanced apoptosis was associated with the decreased m6A level, leading to downregulation of PI3K/AKT protein, ultimately activating the apoptosis-related proteins. Unexpectedly, although combination therapy resulted in metabolic recombination, no significant change in methionine metabolic metabolites was found. More importantly, the combination also exerted synergistic effects in vivo. In summary, the combination of MAT2A inhibitor and METTL3 inhibitor showed synergistic effects both in vivo and in vitro, which laid a theoretical foundation for expanding the clinical application research of the two types of drugs.

Characterization of methionine dependence in melanoma cells.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of S-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

Hematopoietic Stem/Progenitor Cell Intrinsic SAM Synthesis Is Required to Prevent p53 Pathway Activation By Maintaining DNA Stability

S-adenosylmethionine (SAM) is a principle methyl group donor to maintain epigenetic status. SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a (Methionine adenosyltransferase 2a). It has been reported that SAM reduction by methionine restriction or Mat2a inhibition suppresses the proliferation of leukemia cells to attract attentions as a potential therapeutic target for leukemia. To develop the therapy targeting methionine metabolism in leukemia, it is essential to elucidate the significance of methionine metabolism in non-leukemic hematopoiesis. However, still little is known about the importance of SAM synthesis in hematopoiesis, including hematopoietic stem/progenitor cells (HSPCs), in vivo. By using Mx1-Cre mediated Mat2a gene deletion in mice ( Mat2a fl/fl;Mx1-Cre mice), we investigated the roles of Mat2a in hematopoiesis in vivo. Reduction of Mat2a activity in Mat2a knockout (KO) mice in bone marrow (BM) was confirmed by ultra-high performance liquid chromatography tandem mass spectrometry analysis. Mat2a KO mice displayed severe pancytopenia in peripheral blood (PB). Mature cell fractions (Myeloid cells, B cells and T cells) and HSPCs (HSCs, MPP2s, MPP3s and MPP4s) in BM were also significantly decreased in Mat2a KO mice, suggesting that Mat2a is essential for the maintenance of hematopoiesis from HSPCs in vivo. To exclude the possible contributions of the effects induced by Mat2a KO in non-hematopoietic cells, we performed BM transplantation analyses in which hematopoietic cell specific KO or non-hematopoietic cell specific KO were achieved. These analyses clearly confirmed that HSPC intrinsic SAM synthesis is inevitable for the maintenance of hematopoiesis. To reveal the mechanistic insights of the regulatory roles for Mat2a in HSPCs, we performed RNA sequencing analysis ( Mat2a WT vs KO in HSPCs). GSEA (Gene Set Enrichment Analysis) using the Hallmark gene sets showed that p53 pathway was most highly enriched in Mat2a KO. In depth, genes downstream of p53 ( Bax, Apaf1, Bbc3, Casp3, Casp8, Fas, Cdkn1a etc.) were up-regulated by Mat2a KO, causing cell cycle arrest and apoptosis in HSPCs. On the other hand, gene expressions of the upstream of p53 ( Atr, Atm, Chek1, Chek2 etc.) were unchanged, suggesting that p53 activity was not increased at mRNA expression levels. In line with this, mRNA expression levels of Trp53 was unchanged, but total protein amount of p53 was increased in Mat2a KO HSPCs. Since DNA stability is known as a critical regulator of p53 pathway, we analyzed gH2AX levels as DNA damage marker and found that gH2AX levels were increased in Mat2a KO HSPCs. To be noted, expressions of genes involved in Fanc pathway ( Fanca, Fancc, Fancd2, Fance, Fancg, Fancl, Fanci etc), which is responsible for the DNA maintenance, were broadly down-regulated in Mat2a KO HSPCs at transcriptomic levels. Therefore, the Fanc pathway repression by SAM synthesis inhibition could cause DNA damage and p53 pathway dependent loss of maintenance in Mat2a KO HSPCs. To further understand the importance of SAM synthesis under stress hematopoiesis. We analyzed Mat2a expression changes in HSPCs by irradiation (5.5 Gy/mouse). Intriguingly, Mat2a expression was up-regulated by irradiation. We also found that dietary methionine restriction (MR) in mice prevents its hematopoietic recovery from irradiation stress. To be noted, long term MR (24 weeks) did not show any particular compared to control diet. Therefore, methionine metabolism possesses significant roles at least in the situation where acute hematopoietic recovery is required. Collectively, in this study, we found that cell intrinsic SAM synthesis is required for the maintenance (proliferation and survival) of HSPCs in vivo. SAM might be required for the prevention of DNA damage and p53 pathway activation by supporting Fanc pathway gene expressions. Methionine and/or SAM availability might possess critical roles for the regeneration of non-leukemic hematopoiesis. Further investigation about the significance of SAM synthesis in non-leukemic hematopoiesis is required for the proper establishment of SAM synthesis targeting therapy in cancers.

TMET-20. TARGETING LINKS BETWEEN METHIONINE METABOLISM AND DNA REPAIR TO IMPROVE GBM THERAPY

Abstract Glioblastoma (GBM) is uniformly lethal due to intrinsic resistance to standard of care radiation (RT) and chemotherapy. Recent studies have identified altered cellular metabolism as a key mediator of GBM RT resistance. Methionine uptake is drastically elevated in GBMs compared to normal cells but whether this impacts treatment resistance is uncertain, as is the metabolic fates of methionine once it enters GBM cells. Here, we find that RT acutely increases the levels of methionine-related metabolites in a variety of RT-resistant GBM models. Stable isotope tracing studies further revealed that RT acutely activates methionine to S-adenosyl methionine (SAM) conversion through an active signaling event through DNA damage response. We developed in vivo 13C5 methionine stable isotope tracing techniques to confirm these findings in orthotopic models of GBM. Pharmacological and dietary strategies to block methionine to SAM conversion slowed the DNA damage repair and increased cell death following RT. These effects are selective to GBMs lacking the methionine salvage enzyme methylthioadenosine phosphorylase (MTAP). Pharmacologic and dietary strategies to inhibit SAM synthesis hindered tumor growth in flank and orthotopic in vivo GBM models when combined with RT. In short, these results highlight a new signaling link between DNA damage and SAM synthesis and define the metabolic fates of methionine in GBM in vivo. Inhibiting the RT-induced SAM synthesis slows the DNA damage repair and augments RT efficacy in GBM. This therapeutic strategy provides scope for combining MAT2A inhibitors with RT in GBMs with impaired methionine salvage and spares the adjacent normal brain.

Optimized methyl donor and reduced precursor degradation pathway for seleno-methylselenocysteine production in Bacillus subtilis

BackgroundSeleno-methylselenocysteine (SeMCys) is an effective component of selenium supplementation with anti-carcinogenic potential that can ameliorate neuropathology and cognitive deficits. In a previous study, a SeMCys producing strain of Bacillus subtilis GBACB was generated by releasing feedback inhibition by overexpression of cysteine-insensitive serine O-acetyltransferase, enhancing the synthesis of S-adenosylmethionine as methyl donor by overexpression of S-adenosylmethionine synthetase, and expressing heterologous selenocysteine methyltransferase. In this study, we aimed to improve GBACB SeMCys production by synthesizing methylmethionine as a donor to methylate selenocysteine and by inhibiting the precursor degradation pathway.ResultsFirst, the performance of three methionine S-methyltransferases that provide methylmethionine as a methyl donor for SeMCys production was determined. Integration of the NmMmt gene into GBACB improved SeMCys production from 20.7 to 687.4 μg/L. Next, the major routes for the degradation of selenocysteine, which is the precursor of SeMCys, were revealed by comparing selenocysteine hyper-accumulating and non-producing strains at the transcriptional level. The iscSB knockout strain doubled SeMCys production. Moreover, deleting sdaA, which is responsible for the degradation of serine as a precursor of selenocysteine, enhanced SeMCys production to 4120.3 μg/L. Finally, the culture conditions in the flasks were optimized. The strain was tolerant to higher selenite content in the liquid medium and the titer of SeMCys reached 7.5 mg/L.ConclusionsThe significance of methylmethionine as a methyl donor for SeMCys production in B. subtilis is reported, and enhanced precursor supply facilitates SeMCys synthesis. The results represent the highest SeMCys production to date and provide insight into Se metabolism.

Phage T3 overcomes the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase

Bacteriophage T3 encodes a SAMase that, through cleavage of S-adenosyl methionine (SAM), circumvents the SAM-dependent type I restriction-modification (R-M) defense. We show that SAMase also allows T3 to evade the BREX defense. Although SAM depletion weakly affects BREX methylation, it completely inhibits the defensive function of BREX, suggesting that SAM could be a co-factor for BREX-mediated exclusion of phage DNA, similar to its anti-defense role in type I R-M. The anti-BREX activity of T3 SAMase is mediated not just by enzymatic degradation of SAM but also by direct inhibition of MetK, the host SAM synthase. We present a 2.8Å cryoelectron microscopy (cryo-EM) structure of the eight-subunit T3 SAMase-MetK complex. Structure-guided mutagenesis reveals that this interaction stabilizes T3 SAMase invivo, further stimulating its anti-BREX activity. This work provides insights in the versatility of bacteriophage counterdefense mechanisms and highlights the role of SAM as a co-factor of diverse bacterial immunity systems.

Influence of Cobalt Source, Folic Acid, and Rumen-Protected Methionine on Performance, Metabolism, and Liver Tissue One-Carbon Metabolism Biomarkers in Peripartal Holstein Cows.

Vitamin B12 plays a role in the remethylation of homocysteine to Met, which then serves as a substrate for Met adenosyltransferase (MAT) to synthesize S-adenosylmethionine (SAM). We investigated effects of feeding two cobalt sources [Co-glucoheptonate (CoPro) or CoPectin, Zinpro Corp.], an experimental ruminally-available source of folic acid (FOA), and rumen-protected Met (RPM) on performance and hepatic one-carbon metabolism in peripartal Holstein cows. From -30 to 30 d around calving, 72 multiparous cows were randomly allocated to: CoPro, CoPro + FOA, CoPectin + FOA, or CoPectin + FOA + RPM. The Co treatments delivered 1 mg Co/kg of DM (CoPro or CoPectin), each FOA group received 50 mg/d FOA, and RPM was fed at 0.09% of DM intake (DMI). Milk yield and DMI were not affected. Compared with other groups, the percentage of milk protein was greater after the second week of lactation in CoPectin + FOA + RPM. Compared with CoPro or CoPro + FOA, feeding CoPectin + FOA or CoPectin + FOA + RPM led to a greater activity of MAT at 7 to 15 d postcalving. For betaine-homocysteine S-methyltransferase, CoPro together with CoPectin + FOA + RPM cows had greater activity at 7 and 15 d than CoPro + FOA. Overall, supplying FOA with CoPectin or CoPectin plus RPM may enhance S-adenosylmethionine synthesis via MAT in the liver after parturition. As such, these nutrients may impact methylation reactions and liver function.

Integrating biochemical and anatomical characterizations with transcriptome analysis to dissect superior stem strength of ZS11 (Brassica napus).

Stem lodging resistance is a serious problem impairing crop yield and quality. ZS11 is an adaptable and stable yielding rapeseed variety with excellent resistance to lodging. However, the mechanism regulating lodging resistance in ZS11 remains unclear. Here, we observed that high stem mechanical strength is the main factor determining the superior lodging resistance of ZS11 through a comparative biology study. Compared with 4D122, ZS11 has higher rind penetrometer resistance (RPR) and stem breaking strength (SBS) at flowering and silique stages. Anatomical analysis shows that ZS11 exhibits thicker xylem layers and denser interfascicular fibrocytes. Analysis of cell wall components suggests that ZS11 possessed more lignin and cellulose during stem secondary development. By comparative transcriptome analysis, we reveal a relatively higher expression of genes required for S-adenosylmethionine (SAM) synthesis, and several key genes (4-COUMATATE-CoA LIGASE, CINNAMOYL-CoA REDUCTASE, CAFFEATE O-METHYLTRANSFERASE, PEROXIDASE) involved in lignin synthesis pathway in ZS11, which support an enhanced lignin biosynthesis ability in the ZS11 stem. Moreover, the difference in cellulose may relate to the significant enrichment of DEGs associated with microtubule-related process and cytoskeleton organization at the flowering stage. Protein interaction network analysis indicate that the preferential expression of several genes, such as LONESOME HIGHWAY (LHW), DNA BINDING WITH ONE FINGERS (DOFs), WUSCHEL HOMEOBOX RELATED 4 (WOX4), are related to vascular development and contribute to denser and thicker lignified cell layers in ZS11. Taken together, our results provide insights into the physiological and molecular regulatory basis for the formation of stem lodging resistance in ZS11, which will greatly promote the application of this superior trait in rapeseed breeding.

Abstract 1095: Exploiting altered methionine metabolism to overcome treatment resistance in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive adult brain tumor and is uniformly fatal due to resistance to standard therapies such as radiation (RT) and chemotherapy. Our group and others have identified altered metabolism as a key mediator of GBM RT resistance. Methionine is an essential sulfur-containing amino acid that cells use to synthesize antioxidants, polyamines and S-adenosyl methionine (SAM), which drives intracellular methylation reactions. Methionine uptake is dramatically elevated in GBM compared to normal brain, but what GBMs use this methionine for, and whether it governs GBM treatment resistance, is unknown. Here, we find that RT acutely increases the levels of numerous methionine-related metabolites in multiple RT-resistant GBM models. To interrogate metabolic pathway activity, we used 13C5 methionine stable isotope tracing to show that GBMs respond to RT by activating the conversion of methionine to SAM, which is dependent on signaling through the DNA damage response. We developed in vivo methionine stable isotope tracing techniques to confirm these findings in orthotopic PDX models of GBM. Blocking the conversion of methionine to SAM, through pharmacologic inhibition of methionine adenosyltransferase 2A (MAT2A), slowed the repair of RT-induced DNA damage and increased cell death in GBM models following RT. These effects were especially pronounced in GBM models lacking the methionine salvage enzyme methylthioadenosine phosphorylase (MTAP). Pharmacologic inhibition of MAT2A in flank and orthotopic in vivo GBM models depleted SAM levels and slowed tumor growth when combined with RT. Combining MAT2A inhibition with dietary methionine restriction and RT slowed GBM tumor growth even further. Together, our work demonstrates a new signaling link between DNA damage and methionine-driven SAM synthesis in GBM. Inhibiting SAM synthesis slows the repair of RT-induced DNA damage and augments RT efficacy. This therapeutic strategy may be especially effective in GBMs defective in methionine salvage and spare normal cortex in which methionine salvage is active. Citation Format: Navyateja Korimerla, Kari-Wilder Romans, Peter Kalev, Ayesha Kothari, Nathan Qi, Charles Evans, Maureen Kachman, Marc L Hyer, Katya Marjon, Taryn Sleger, Daniel R Wahl. Exploiting altered methionine metabolism to overcome treatment resistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1095.

Abstract 6045: Role of PP2A methylation on methionine dependence of cancer

Abstract Dependence on exogenous methionine is a well-known, but poorly understood cancer-specific metabolic requirement. Although most cancer cells can convert homocysteine to methionine, they are unable to grow when methionine depleted media is supplemented with homocysteine. In contrast, non-tumorigenic cells proliferate well in homocysteine media. Previous work from our laboratory has shown that cancer cells cultured in homocysteine medium increase metabolic flux towards the transsulfuration pathway, away from S-adenosylmethionine (SAM) synthesis resulting in overall reduced methylation potential. We have shown that methionine sensing occurs independently of the mTOR pathway, which is known to link amino acid abundance and cell growth. Instead, our results suggest that Protein Phosphatase 2A (PP2A) methylation is sensitive to changes in the SAM/SAH ratio. Reduced PP2A methylation correlates with reduced cell proliferation in homocysteine medium. Furthermore, overexpression of the PP2A demethylase Pme1 forcibly reduces PP2A methylation and induces methionine dependence in cells that normally proliferate in homocysteine medium. These findings suggest a model that links methionine metabolism to cell cycle progression via regulation of PP2A methylation. Here we use quantitative proteomics to define changes in B-subunit binding to PP2A during methionine starvation to investigate the molecular mechanism of this methionine dependence seen in cancer. Citation Format: Anna Andronicos, Da-Wei Lin, Yi-Chen Su, Mari Ishak Gabra, Mei Kong, Peter Kaiser. Role of PP2A methylation on methionine dependence of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6045.

Abstract 1644: Dual inhibition of MAT2A and PRMT5 delivers synergistic anti-tumor responses in preclinical models of MTAP-deleted cancer

Abstract Approximately 15% of all tumors harbor deletions in the 9p21 locus that encompass CDKN2A and MTAP. MTAP fuels the adenine and methionine salvage pathways by converting MTA into precursors of building blocks for protein and DNA production. In MTAP-deleted (MTAPdel) tumors, cellular MTA accumulates and partially inhibits the essential protein methyl transferase, PRMT5, through direct displacement of the cofactor S-adenosyl methionine (SAM) from the PRMT5 active site. This context provides therapeutic opportunities, currently under clinical evaluation, to selectively extinguish PRMT5 activity in tumor cells with MAT2A inhibitors that limit SAM synthesis or with direct PRMT5 inhibitors that are MTA-cooperative. Preclinical modeling of these synthetic lethal relationships, and elucidation of their cellular mechanisms of action, suggests efficacy can vary depending upon the extent of MTA accumulation and/or adaptive compensation to PRMT5 inhibition. To potentially maximize therapeutic activity among mechanistically heterogenous MTAPdel tumors, we evaluated combinatorial pathway suppression with MAT2A and PRMT5 inhibitors in MTAPWT and MTAPdel tumor models in vitro and in vivo. We noted synergistic antiproliferative effects when the potent and selective MAT2A inhibitor IDE397 was combined with any of multiple MTA-cooperative PRMT5 inhibitors in MTAPdel cell lines that exhibit sensitivity to each single agent. In vivo, the combination of IDE397 and MTA-cooperative PRMT5 inhibitors were well tolerated, and induced durable tumor regressions, including complete responses, at dose levels well below the maximally efficacious preclinical dose of each individual agent in MTAPdel lung adenocarcinoma and pancreas cancer models H838 and BXPC3. Pharmacodynamic target engagement in the tumor was evaluated by quantitative assessment of symmetric dimethyl arginine (SDMA), as detected by IHC, and indicated earlier onset and greater extent of PRMT5 inhibition by combination treatment as compared to either agent alone. The potentiation of the antitumor response with the combination did not occur in the MTAPWT setting. Thus, combined inhibition of MAT2A and PRMT5 potentially offers a compelling dual synthetic lethal opportunity to address unmet need for the many patients afflicted with MTAPdel cancers. Citation Format: Marcus M. Fischer, Kimberline Gerrick, Brian Belmontes, Katherine Slemmons, Yevgeniy Freyman, Jay Jain, Steve Federowicz, Isaac Bishof, Arjun A. Rao, Melissa Fleury, Zineb Mounir, Mark R. Lackner, Paul E. Hughes, Mike White, Claire L. Neilan. Dual inhibition of MAT2A and PRMT5 delivers synergistic anti-tumor responses in preclinical models of MTAP-deleted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1644.

α-Lipoic acid eliminates dioxin-induced offspring sexual immaturity by improving abnormalities in folic acid metabolism

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes developmental and reproductive disorders in pups due to the attenuated luteinizing hormone (LH) production during the perinatal stage; however, the administration of α-lipoic acid (LA) to TCDD-exposed pregnant rats reversed the attenuated LH production. Therefore, reproductive disorders in pups are expected to be ameliorated with LA supplementation. To address this issue, pregnant rats orally received low dose TCDD at gestational day 15 (GD15) and proceeded to parturition. The control received a corn oil vehicle. To examine the preventive effects of LA, supplementation with LA was provided until postnatal day 21. In this study, we demonstrated that maternal administration of LA restored the sexually dimorphic behavior of male and female offspring. TCDD-induced LA insufficiency is likely a direct cause of TCDD reproductive toxicity. In the analysis to clarify the mechanism of the decrease in LA, we found evidence suggesting that TCDD inhibits the synthesis and increases the utilization of S-adenosylmethionine (SAM), a cofactor for LA synthesis, resulting in a decrease in the SAM level. Furthermore, folate metabolism, which is involved in SAM synthesis, is disrupted by TCDD, which may adversely affect infant growth. Maternal supplementation of LA restored SAM to its original level in the fetal hypothalamus; in turn, SAM ameliorated abnormal folate consumption and suppressed aryl hydrocarbon receptor activation induced by TCDD. The study demonstrates that the application of LA could prevent and recover next-generation dioxin reproductive toxicity, which provides the potential to establish effective protective measures against dioxin toxicity.

Hepatic oleate regulates one-carbon metabolism during high carbohydrate feeding

Obesity is a major risk factor for type 2 diabetes, coronary heart disease, and strok. These diseases are associated with profound alterations in gene expression in metabolic tissues. Epigenetic-mediated regulation of gene expression is one mechanism through which environmental factors, such as diet, modify gene expression and disease predisposition. However, epigenetic control of gene expression in obesity and insulin resistance is not fully characterized. We discovered that liver-specific stearoyl-CoA desaturase-1 (Scd1) knockout mice (LKO) fed a high-carbohydrate low-fat diet exhibit dramatic changes in hepatic gene expression and metabolites of the folate cycle and one-carbon metabolism respectively for the synthesis of S-adenosylmethionine (SAM). LKO mice show an increased ratio of S-adenosylmethionine to S-adenosylhomocysteine, a marker for increased cellular methylation capacity. Furthermore, expression of DNA and histone methyltransferase genes is up-regulated while the mRNA and protein levels of the non-DNA methyltransferases including phosphatidylethanolamine methyltransferase (PEMT), Betaine homocysteine methyltransferase (Bhmt), and the SAM-utilizing enzymes such as glycine-N-methyltransferase (Gnmt) and guanidinoacetate methyltransferase (Gamt) are generally down-regulated. Feeding LKO mice a high carbohydrate diet supplemented with triolein, but not tristearin, and increased endogenous hepatic synthesis of oleate but not palmitoleate in Scd1 global knockout mice normalized one carbon gene expression and metabolite levels. Additionally, changes in one carbon gene expression are independent of the PGC-1α-mediated ER stress response previously reported in the LKO mice. Together, these results highlight the important role of oleate in maintaining one-carbon cycle homeostasis and point to observed changes in one-carbon metabolism as a novel mediator of the Scd1 deficiency-induced liver phenotype.

Lack of Association of C677T Methylenetetrahydrofolate Reductase Polymorphism with Breast Cancer Risk in Mali.

Methylenetetrahydrofolate reductase (MTHFR) plays a major role in the metabolism of folates and homocysteine, which in turn can affect gene expression and ultimately promote the development of breast cancer. Thus, mutations in the MTHFR gene could influence homocysteine, methionine, and S-adenosylmethionine levels and, indirectly, nucleotide levels. Imbalance in methionine and S-adenosylmethionine synthesis affects protein synthesis and methylation. These changes, which affect gene expression, may ultimately promote the development of breast cancer. We therefore hypothesized that such mutations could also play an important role in the occurrence and pathogenesis of breast cancer in a Malian population. In this study, we used the PCR-RFLP technique to identify the different genotypic profiles of the C677T MTHFR polymorphism in 127 breast cancer women and 160 healthy controls. The genotypic distribution of the C677T polymorphism in breast cancer cases was 88.2% for CC, 11.0% for CT, and 0.8% for TT. Healthy controls showed a similar distribution with 90.6% for CC, 8.8% for CT, and 0.6% for TT. We found no statistical association between the C677T polymorphism and breast cancer risk for the codominant models CT and TT (p > 0.05). The same trend was observed when the analysis was extended to other genetic models, including dominant (p = 0.50), recessive (p = 0.87), and additive (p = 0.50) models. The C677T polymorphism of MTHFR gene did not influence the risk of breast cancer in the Malian samples.

Circ-ERC2 Is Involved in Melatonin Synthesis by Regulating the miR-125a-5p/MAT2A Axis

The circadian rhythm of melatonin secretion in the pineal gland is highly conserved in vertebrates. Melatonin levels are always elevated at night. Acetylserotonin O-methyltransferase (ASMT) is the last enzyme in the regulation of melatonin biosynthesis (N-acetyl-5-hydroxytryptamine-melatonin). S-adenosylmethionine (SAM) is an important methyl donor in mammals and can be used as a substrate for the synthesis of melatonin. Methionine adenosyltransferase (MAT) catalyzes the synthesis of SAM from methionine and ATP and has a circadian rhythm. CircRNA is an emerging type of endogenous noncoding RNA with a closed loop. Whether circRNAs in the pineal gland can participate in the regulation of melatonin synthesis by binding miRNAs to target mat2a as part of the circadian rhythm is still unclear. In this study, we predicted the targeting relationship of differentially expressed circRNAs, miRNAs and mRNAs based on the results of rat pineal RNA sequencing. Mat2a siRNA transfection confirmed that mat2a is involved in the synthesis of melatonin. Circ-ERC2 and miR-125a-5p were screened out by software prediction, dual-luciferase reporter experiments, cell transfection, etc. Finally, we constructed a rat superior cervical ganglionectomy model (SCGx), and the results showed that circ-ERC2 could participate in the synthesis of melatonin through the miR-125a-5p/MAT2A axis. The results of the study revealed that circ-ERC2 can act as a molecular sponge of miR-125a-5p to regulate the synthesis of melatonin in the pineal gland by targeting mat2a. This experiment provides a basis for research on the circadian rhythm of noncoding RNA on pineal melatonin secretion.

Stemness of Normal and Cancer Cells: The Influence of Methionine Needs and SIRT1/PGC-1α/PPAR-α Players.

Stem cells are a population of undifferentiated cells with self-renewal and differentiation capacities. Normal and cancer stem cells share similar characteristics in relation to their stemness properties. One-carbon metabolism (OCM), a network of interconnected reactions, plays an important role in this dependence through its role in the endogenous synthesis of methionine and S-adenosylmethionine (SAM), the universal donor of methyl groups in eukaryotic cells. OCM genes are differentially expressed in stem cells, compared to their differentiated counterparts. Furthermore, cultivating stem cells in methionine-restricted conditions hinders their stemness capacities through decreased SAM levels with a subsequent decrease in histone methylation, notably H3K4me3, with a decrease in stem cell markers. Stem cells' reliance on methionine is linked to several mechanisms, including high methionine flux or low endogenous methionine biosynthesis. In this review, we provide an overview of the recent discoveries concerning this metabolic dependence and we discuss the mechanisms behind them. We highlight the influence of SIRT1 on SAM synthesis and suggest a role of PGC-1α/PPAR-α in impaired stemness produced by methionine deprivation. In addition, we discuss the potential interest of methionine restriction in regenerative medicine and cancer treatment.

Metabolic regulation by p53 prevents R-loop-associated genomic instability.

Gene-environment interactions can perturb the epigenome, triggering network alterations that participate in cancer pathogenesis. Integrating epigenomics, transcriptomics, and metabolic analyses with functional perturbation, we show that the tumor suppressor p53 preserves genomic integrity by empowering adequate levels of the universal methyl donor S-adenosylmethionine (SAM). In p53-deficient cells, perturbation of DNA methylation promotes derepression of heterochromatin, massive loss of histone H3-lysine 9 methylation, and consequent upregulation of satellite RNAs that triggers R-loop-associated replication stress and chromosomal aberrations. In p53-deficient cells, the inadequate SAM level underlies the inability to respond to perturbation because exogenous reintroduction of SAM represses satellite elements and restores the ability to cope with stress. Mechanistically, p53 transcriptionally controls genes involved in one-carbon metabolism, including Slc43a2, the methionine uptake transporter that is critical for SAM synthesis. Supported by clinical data, our findings shed light on the role of p53-mediated metabolism in preventing unscheduled R-loop-associated genomic instability.

One Carbon Metabolism and S-Adenosylmethionine in Non-Alcoholic Fatty Liver Disease Pathogenesis and Subtypes.

One carbon metabolism (1CM) can be defined as the transfer of a carbon unit from one metabolite to another and its replenishment by different sources of labile methyl-group nutrients: primarily choline, methionine, betaine, and serine. This flow of carbon units allows the biosynthesis of nucleotides, amino acids, formylated methionyl-tRNA, polyamines, glutathione, phospholipids, detoxification reactions, maintenance of the redox status and the concentration of NAD, and methylation reactions including epigenetic modifications. That is, 1CM functions as a nutrient sensor and integrator of cellular metabolism. A critical process in 1CM is the synthesis of S-adenosylmethionine (SAMe), the source of essentially all the hundreds of millions of daily methyl transfer reactions in a cell. This versatility of SAMe imposes a tight control in its synthesis and catabolism. Much of our knowledge concerning 1CM has been gained from studies in the production and prevention of nonalcoholic fatty liver disease (NAFLD). Here, we discuss in detail the function of the most important enzymes for their quantitative contribution to maintaining the flux of carbon units through 1CM in the liver and discuss how alterations in their enzymatic activity contribute to the development of NAFLD. Next, we discuss NAFLD subtypes based on serum lipidomic profiles with different risk of cardiovascular disease. Among the latter, we highlight the so-called subtype A for its serum lipidomic profile phenocopying that of mice deficient in SAMe synthesis and because its high frequency (about 50% of the NAFLD patients).