Cellular S-adenosylmethionine Research Articles

Modulation of m6A RNA modification by DAP3 in cancer cells

N6-methyladenosine (m6A) RNA methylation is a prevalent RNA modification that significantly impacts RNA metabolism and cancer development. Maintaining the global m6A levels in cancer cells relies on RNA accessibility to methyltransferases and the availability of the methyl donor S-adenosylmethionine (SAM). Here, we reveal that death associated protein 3 (DAP3) plays a crucial role in preserving m6A levels through two distinct mechanisms. First, although DAP3 is not a component of the m6A writer complex, it directly binds to m6A target regions, thereby facilitating METTL3 binding. Second, DAP3 promotes MAT2A's last intron splicing, increasing MAT2A protein, cellular SAM, and m6A levels. Silencing DAP3 hinders tumorigenesis, which can be rescued by MAT2A overexpression. This evidence suggests DAP3's role in tumorigenesis, partly through m6A regulation. Our findings unveil DAP3's complex role as an RNA-binding protein and tumor promoter, impacting RNA processing, splicing, and m6A modification in cancer transcriptomes.

Targeting protein methylation in pancreatic cancer cells results in KRAS signaling imbalance and inhibition of autophagy

Pancreatic cancer cells with mutant KRAS require strong basal autophagy for viability and growth. Here, we observed that some processes that allow the maintenance of basal autophagy in pancreatic cancer cells are controlled by protein methylation. Thus, by maintaining the methylation status of proteins such as PP2A and MRAS, these cells can sustain their autophagic activity. Protein methylation disruption by a hypomethylating treatment (HMT), which depletes cellular S-adenosylmethionine levels while inducing S-adenosylhomocysteine accumulation, resulted in autophagy inhibition and endoplasmic reticulum stress-induced apoptosis in pancreatic cancer cells. We observed that by reducing the membrane localization of MRAS, hypomethylation conditions produced an imbalance in KRAS signaling, resulting in the partial inactivation of ERK and hyperactivation of the PI3K/AKT–mTORC1 pathway. Interestingly, HMT impeded CRAF activation by disrupting the ternary SHOC2 complex (SHOC2/MRAS/PP1), which functions as a CRAF-S259 holophosphatase. The demethylation events that resulted in PP2A inactivation also favored autophagy inhibition by preventing ULK1 activation while restoring the cytoplasmic retention of the MiT/TFE transcription factors. Since autophagy provides pancreatic cancer cells with metabolic plasticity to cope with various metabolic stress conditions, while at the same time promoting their pathogenesis and resistance to KRAS pathway inhibitors, this hypomethylating treatment could represent a therapeutic opportunity for pancreatic adenocarcinomas.

Intrinsic catalytic properties of histone H3 lysine-9 methyltransferases preserve monomethylation levels under low S-adenosylmethionine

S-adenosylmethionine (SAM) is the methyl donor for site-specific methylation reactions on histone proteins, imparting key epigenetic information. During SAM-depleted conditions that can arise from dietary methionine restriction, lysine di- and tri-methylation are reduced while sites such as Histone-3 lysine-9 (H3K9) are actively maintained, allowing cells to restore higher-state methylation upon metabolic recovery. Here, we investigated if the intrinsic catalytic properties of H3K9 histone methyltransferases (HMTs) contribute to this epigenetic persistence. We employed systematic kinetic analyses and substrate binding assays using four recombinant H3K9 HMTs (i.e., EHMT1, EHMT2, SUV39H1, and SUV39H2). At both high and low (i.e., sub-saturating) SAM, all HMTs displayed the highest catalytic efficiency (kcat/KM) for monomethylation compared to di- and trimethylation on H3 peptide substrates. The favored monomethylation reaction was also reflected in kcat values, apart from SUV39H2 which displayed a similar kcat regardless of substrate methylation state. Using differentially methylated nucleosomes as substrates, kinetic analyses of EHMT1 and EHMT2 revealed similar catalytic preferences. Orthogonal binding assays revealed only small differences in substrate affinity across methylation states, suggesting that catalytic steps dictate the monomethylation preferences of EHMT1, EHMT2, and SUV39H1. To link invitro catalytic rates with nuclear methylation dynamics, we built a mathematical model incorporating measured kinetic parameters and a time course of mass spectrometry-based H3K9 methylation measurements following cellular SAM depletion. The model revealed that the intrinsic kinetic constants of the catalytic domains could recapitulate invivo observations. Together, these results suggest catalytic discrimination by H3K9 HMTs maintains nuclear H3K9me1, ensuring epigenetic persistence after metabolic stress.

DIPG-36. H3K27M HISTONE MUTANT GLIOMAS ARE SENSITIVE TO METHIONINE LOSS AND MAT2A INHIBITION THROUGH A NOVEL FEEDBACK MECHANISM BETWEEN AMD1 AND METTL16

Abstract Diffuse Midline gliomas (DMGs) are grade IV tumors by the World Health Organization. They are inoperable and resistant to chemo/radiotherapies resulting in a median survival of 8-11 months and a 5-year survival of <2%. DMG is an epigenetic disease characterized by mutations on histone H3.3 K27M resulting in global transcriptional reprogramming. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model using clinically relevant co-alterations in Olig2+ neural progenitor cells (NPCs). Using an unbiased systems biology approach, we identified a reliance of H3K27M but not isogenic controls to the amino acid methionine, and the enzymes methionine adenosyltransferase 2A (MAT2A), and adenosylmethionine decarboxylase 1 (AMD1). MAT2A is a master regulator of methionine metabolism that converts methionine into the universal methyl donor S-adenosylmethionine (SAM) which is later converted into decarboxylated SAM (dcSAM) by AMD1 for polyamine metabolism. We postulated that targeting methionine regulator MAT2A through genetic/pharmacological abrogation would selectively alter DMG viability by disrupting the methylome. We discovered a novel mechanism demonstrating H3K27M cells are sensitive to MAT2A loss independent of methylthioadenosine phosphorylase (MTAP) deletions but rather through AMD1 overexpression. The current paradigm shows that MAT2A protein expression is inversely correlated with cellular SAM concentrations as sensed by splicing complex and m6A reader methyltransferase-like protein 16 (METTL16). To investigate the molecular mechanism by which H3K27M represses MAT2A, we postulated that dcSAM, the resultant metabolite of AMD1, promote(s) high turnover of METTL16–MAT2A transcript interactions like SAM, thereby diminishing MAT2A transcript and protein expression. We found that exogenous dcSAM promoted MAT2A intron retention and lower mature transcript levels. Our findings demonstrate that H3K27M leads to increased AMD1 protein expression resulting in diminished MAT2A expression. Combinatorial treatments inhibiting MAT2A and AMD1 may presents exploitable therapeutic vulnerabilities in these gliomas.

Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids

Methionine restriction (MR) provides metabolic benefits in many organisms. However, mechanisms underlying the MR-induced effect remain incompletely understood. Here, we show in the budding yeast S. cerevisiae that MR relays a signal of S-adenosylmethionine (SAM) deprivation to adapt bioenergetic mitochondria to nitrogenic anabolism. In particular, decreases in cellular SAM constrain lipoate metabolism and protein lipoylation required for the operation of the tricarboxylic acid (TCA) cycle in the mitochondria, leading to incomplete glucose oxidation with an exit of acetyl-CoA and α-ketoglutarate from the TCA cycle to the syntheses of amino acids, such as arginine and leucine. This mitochondrial response achieves a trade-off between energy metabolism and nitrogenic anabolism, which serves as an effector mechanism promoting cell survival under MR.

MAT Gain of Activity Mutation in Helicobacter pylori Is Associated with Resistance to MTAN Transition State Analogues.

Helicobacter pylori is found in the gut lining of more than half of the world's population, causes gastric ulcers, and contributes to stomach cancers. Menaquinone synthesis in H. pylori relies on the rare futalosine pathway, where H. pylori 5'-methylthioadenosine nucleosidase (MTAN) is proposed to play an essential role. Transition state analogues of MTAN, including BuT-DADMe-ImmA (BTDIA) and MeT-DADMe-ImmA (MTDIA), exhibit bacteriostatic action against numerous diverse clinical isolates of H. pylori with minimum inhibitory concentrations (MIC's) of <2 ng/mL. Three H. pylori BTDIA-resistant clones were selected under increasing BTDIA pressure. Whole genome sequencing showed no mutations in MTAN. Instead, resistant clones had mutations in metK, methionine adenosyltransferase (MAT), feoA, a regulator of the iron transport system, and flhF, a flagellar synthesis regulator. The mutation in metK causes expression of a MAT with increased catalytic activity, leading to elevated cellular S-adenosylmethionine. Metabolite analysis and the mutations associated with resistance suggest multiple inputs associated with BTDIA resistance. Human gut microbiome exposed to MTDIA revealed no growth inhibition under aerobic or anaerobic conditions. Transition state analogues of H. pylori MTAN have potential as agents for treating H. pylori infection without disruption of the human gut microbiome or inducing resistance in the MTAN target.

A Phase 1 Dose-Escalation Study of the Cladribine Added to CPX-351 in Patients with Relapsed/Refractory Acute Myeloid Leukemia (AML) a Study of the Polish Adult Leukemia Group (AML-1/2018): Preliminary Results of the Dose Escalation Stage

Two first authors equally contributed to the study Introduction: CPX-351, a liposomal formula of cytarabine (AraC) and daunorubicine (DNR), was shown as a highly potent drug in newly diagnosed high-risk/secondary acute myeloid leukemia (AML) (Lancet JE, J Clin Oncol 2018). Cladribine is a purine analogue that acts by increasing cellular uptake of AraC in leukemic blasts as well as by direct inhibition of DNA synthesis (Robak T, Leuk Res 2014). Additionally, cladribine compromises DNA methylation in leukemic cells by affecting the cellular S-adenosylmethionine (SAM) level (Libura M, et al. Sci Rep. 2021). Based on the favorable results of cladribine-based salvage regimens (CLAG-M, CLAG) (Wierzbowska A; Eur J Hematol 2008) and induction DAC therapy (Hołowiecki J, J Clin Oncol 2012; Pluta A, Am J Hematology 2017), the phase I/II study was designed to explore safety (MTD), toxicity and efficacy of increasing doses of cladribine in combination with standard dose of CPX-351 in relapsed/refractory AML (R/R AML) patients (pts). (EudraCT number: 2020-002535-29). Here we present the results of the first three three-participant cohorts of patients enrolled. Methods: Pts with R/R AML were eligible if age ≥55 years, ECOG performance status ≤1, HCT-CI ≤3, left ventricular ejection fraction (LVEF) by echocardiogram >50%, adequate hepatic and renal function, and no prior CPX-351 therapy. CPX-351 induction therapy was administered by 90-minute infusion at a dose of 100 U/m2 on days 1, 3, and 5. Cladribine dose escalation started at 1 mg/m2, followed by 3 mg/m2 and 5 mg/m2 in 2 hours intravenous infusion, 2 hours before CPX-351 in 3+3 design. DLTs were assessed in cycle 1. Disease response was assessed by the 2017 ELN criteria. Allogeneic hematopoietic cell transplantation (allo-HCT) was recommended for all eligible pts who had a donor immediately after achieving a complete remission (CR). Patients not eligible for transplant received up to 2 consolidation cycles of CPX-351 + cladribine at the same dose. Results: Nine pts were enrolled with a median age 65.5 (range 62-68) years and 55% of pts were male. Pts were generally heavily pretreated, with a median of 2 prior therapies (range 1-4), importantly 78% (7) pts were primary refractory to the former treatment. Fifty percent of pts were in the poor risk group according to ELN2017. One patient (11%) had previous allo-HCT. No DLTs have been observed to date. No drug-related serious adverse events (SAEs) were reported in cohort 1. Also, in cohort 2 there were no drug-related SAE reports. In cohort 3, till now one death potentially related to treatment was reported. It was due to a septic shock during G4 neutropenia. Two other pts are still in aplasia after the first induction cycle. To date, toxicities of grade ≥ 3 during the induction cycle were: febrile neutropenia (88%), infection (88%), neutropenic enterocolitis (11%), and vulvitis (11%). In cohorts 1 and 2, 50% (3) AML pts achieved a CR/CRi, one partial response (PR), and 2 not responded (NR). In cohort 3, assessment of treatment response is not available yet, however, 2 pts were evaluable for early response evaluation on day 14 and both have the blast clearancein the bone marrow. The third patient died early in aplasia. From cohorts 1 and 2, 3 (50%) of pts were transplanted. Conclusions: Preliminary results suggest that cladribine at doses 1 and 3mg/m2 administered with a standard dose of CPX-351 is well tolerated with no DLTs observed. The dose 5mg/m2 is still under evaluation. Till now the safety profile does not indicate any significant additional myelosuppression. CR was observed in 50% of heavily pretreated R/R AML pts indicating satisfying clinical activity. Further evaluation of the safety and efficacy of this regimen is ongoing.

Implications of trimethylamine N-oxide (TMAO) and Betaine in Human Health: Beyond Being Osmoprotective Compounds.

Osmolytes are naturally occurring small molecular weight organic molecules, which are accumulated in large amounts in all life forms to maintain the stability of cellular proteins and hence preserve their functions during adverse environmental conditions. Trimethylamine N-oxide (TMAO) and N,N,N-trimethylglycine (betaine) are methylamine osmolytes that have been extensively studied for their diverse roles in humans and have demonstrated opposing relations with human health. These osmolytes are obtained from food and synthesized endogenously using dietary constituents like choline and carnitine. Especially, gut microbiota plays a vital role in TMAO synthesis and contributes significantly to plasma TMAO levels. The elevated plasma TMAO has been reported to be correlated with the pathogenesis of numerous human diseases, including cardiovascular disease, heart failure, kidney diseases, metabolic syndrome, etc.; Hence, TMAO has been recognized as a novel biomarker for the detection/prediction of several human diseases. In contrast, betaine acts as a methyl donor in one-carbon metabolism, maintains cellular S-adenosylmethionine levels, and protects the cells from the harmful effects of increased plasma homocysteine. Betaine also demonstrates antioxidant and anti-inflammatory activities and has a promising therapeutic value in several human diseases, including homocystinuria and fatty liver disease. The present review examines the multifarious functions of TMAO and betaine with possible molecular mechanisms towards a better understanding of their emerging and diverging functions with probable implications in the prevention, diagnosis, and treatment of human diseases.

RNA methyltransferase METTL16: Targets and function.

The N6‐methyladenosine (m6A) RNA methyltransferase METTL16 is an emerging player in the RNA modification landscape of the human cell. Originally thought to be a ribosomal RNA methyltransferase, it has now been shown to bind and methylate the MAT2A messenger RNA (mRNA) and U6 small nuclear RNA (snRNA). It has also been shown to bind the MALAT1 long noncoding RNA and several other RNAs. METTL16's methyltransferase domain contains the Rossmann‐like fold of class I methyltransferases and uses S‐adenosylmethionine (SAM) as the methyl donor. It has an RNA methylation consensus sequence of UACAGARAA (modified A underlined), and structural requirements for its known RNA interactors. In addition to the methyltransferase domain, METTL16 protein has two other RNA binding domains, one of which resides in a vertebrate conserved region, and a putative nuclear localization signal. The role of METTL16 in the cell is still being explored, however evidence suggests it is essential for most cells. This is currently hypothesized to be due to its role in regulating the splicing of MAT2A mRNA in response to cellular SAM levels. However, one of the more pressing questions remaining is what role METTL16's methylation of U6 snRNA plays in splicing and potentially cellular survival. METTL16 also has several other putative coding and noncoding RNA interactors but the definitive methylation status of those RNAs and the role METTL16 plays in their life cycle is yet to be determined. Overall, METTL16 is an intriguing RNA binding protein and methyltransferase whose important functions in the cell are just beginning to be understood.This article is categorized under:RNA Processing > RNA Editing and ModificationRNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes

Abstract 1956: In vitro and in vivo characterization of novel MAT2A allosteric inhibitors

Abstract Background: MAT2A (Methionine adenosyltransferase 2A) catalyzes the formation of S-adenosylmethionine (SAM) from methionine and ATP. PRMT5, a highly conserved protein arginine methyltransferase that is required for cell proliferation, uses SAM as its methyl donor. PRMT5 activity is inhibited by increased MTA levels in methylthioadenosine phosphorylase (MTAP)-deleted tumor cells. MTAP-deleted tumor cells are thus more vulnerable to MAT2A inhibition compared to MTAP-WT cells. This synthetic lethality between MTAP and MAT2A suggests that MAT2A inhibitors may be useful as anti-tumor agents in the MTAP-deleted cancers, which occurs in ~ 15% of all cancers. Results: We have designed, synthesized and evaluated a novel series of small-molecule MAT2A inhibitors that are very potent in biochemical assays. Co-crystal structures of inhibitor-MAT2A complexes were solved and shed light on binding mode and key interactions. Biochemical mechanistic studies suggested that these compounds are allosteric inhibitors of MAT2A and are non-competitive inhibitors of MAT2A with respect to both ATP and methionine. These compounds displayed potent cellular anti-proliferation activity as well as cellular SAM modulation along with desired DMPK properties. Optimized compounds, upon oral dosing, demonstrated significant PD marker modulation and tumor growth inhibition in HCT116 MTAP-null and several endogenous MTAP-null xenograft mouse models. These agents demonstrated an acceptable safety profile in 7-day oral repeat dose tolerability studies in the rat. Citation Format: Zhonghua Pei, Muzaffar Alam, Neil Bhola, Leah Cleary, Jake Drummond, Marcus Fisher, Melissa Fleury, Candy Garcia, Atieh Givmanesh, Xin Linghu, Ethan McSpadden, Zineb Mounir, Claire Neilan, Zach Newby, Senthil Perumal, Richard Steel, James Sutton, Kedar Vaidya, Marie-Claire Wagle, Jianhong Wang, Bing Yao, Mark Lackner, Michael P. Dillon. In vitro and in vivo characterization of novel MAT2A allosteric inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1956.

A SAM-I riboswitch with the ability to sense and respond to uncharged initiator tRNA

All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.

Metabolic reprogramming of the stromal epigenome in ovarian cancer metastasis

High grade serous ovarian cancer (HGSOC) is characterized by early metastasis throughout the peritoneal cavity and a poor clinical outcome. During the metastatic process, ovarian cancer cells interact with diverse cell populations, including mesothelial cells, adipocytes, and cancer‐associated fibroblasts (CAFs). Ovarian cancer CAFs have well‐described roles in promoting the growth and progression of ovarian cancer by secreting pro‐proliferative cytokines and oncogenic extracellular matrix components (ECM). To better understand the proteomic changes that occur during metastasis in both tumor and stromal compartments, laser‐capture microdissection was coupled to label‐free, quantitative proteomics to independently characterize the proteomes of both tumor and stromal compartments in a cohort of HGSOC patients. We identified a conserved stromal proteomic signature associated with metastasis which included high expression of nicotinamide N‐methyltransferase (NNMT) in the metastatic stroma. NNMT is a metabolic enzyme that catalyzes the methylation of nicotinamide and consumes S‐adenosyl methionine (SAM), thus depleting cellular methylation potential and reducing methylation of DNA and proteins. NNMT regulated the expression of thousands of genes and was necessary and sufficient for the reprogramming of normal fibroblasts to CAFs. Stromal NNMT expression altered multiple metabolic pathways and led to a significant reduction in cellular SAM pools. Attenuated DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) were associated with global changes in gene expression. Using genetic and small‐molecule inhibitor strategies, we found that stromal NNMT activity promoted the progression and metastasis of ovarian cancer. This study provides support to the idea that stromal methyltransferase activities can be targeted to normalize the metastatic stroma and should be further explored as treatment targets for cancer.Support or Funding InformationThis work was supported by a Marsha Rivkin Foundation award (MAE), National Cancer Institute (NCI) grants CA111882 and CA211916 (EL), the Harris Family Foundation (MAE and SDY), the Ludwig Institute for Cancer Research (EL), NIH grant CA175399 and DP2GM128199 (REM), V Foundation for Cancer Research (V2016‐020 to REM), the Körber Foundation/Körber European Science Prize (MM), the Max‐Planck Society for the Advancement of Science (MM), the Novo Nordisk Foundation (Grant agreement NNF14CC0001 and NNF15CC0001; FC and MM), and University of Chicago Cancer Center Support Grant P30CA014599.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of folate and methionine metabolism by multisite phosphorylation of human methylenetetrahydrofolate reductase

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methylene-tetrahydrofolate (THF) to 5-methyl-THF, thereby committing one-carbon units to the methionine cycle. While MTHFR has long been known to be allosterically inhibited by S-adenosylmethionine (SAM), only relatively recently has N-terminal multisite phosphorylation been shown to provide an additional layer of regulation. In vitro, the multiply phosphorylated form of MTHFR is more sensitive to allosteric inhibition by SAM. Here we sought to investigate the kinases responsible for MTHFR multisite phosphorylation and the physiological function of MTHFR phosphorylation in cells. We identified DYRK1A/2 and GSK3A/B among the kinases that phosphorylate MTHFR. In addition, we found that MTHFR phosphorylation is maintained by adequate cellular SAM levels, which are sensed through the C-terminal SAM binding domain of MTHFR. To understand the function of MTHFR phosphorylation in cells, we generated MTHFR CRISPR knockin mutant lines that effectively abolished MTHFR phosphorylation and compared them with the parental cell lines. Whereas the parental cell lines showed increased 5-methyl-THF production in response to homocysteine treatment, the knockin cell lines had high basal levels of 5-methyl-THF and did not respond to homocysteine treatment. Overall, our results suggest that MTHFR multisite phosphorylation coordinates with SAM binding to inhibit MTHFR activity in cells.

Depletion of S-adenosylmethionine impacts on ribosome biogenesis through hypomodification of a single rRNA methylation

S-adenosylmethionine (SAM) is an essential metabolite and a methyl group donor in all living organisms. The intracellular SAM concentration is tightly regulated, and depletion causes hypomethylation of substrates, growth defects and pathological consequences. In the emerging field of epitranscriptomics, SAM-dependent RNA methylations play a critical role in gene expression. Herein, we analyzed the methylation status of ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) in Escherichia coli Δmtn strain in which cellular SAM was down-regulated, and found hypomodification of several methylation sites, including 2′-O-methylation at position 2552 (Um2552) of 23S rRNA. We observed severe growth defect of the Δmtn strain with significant accumulation of 45S ribosomal precursor harboring 23S rRNA with hypomodified Um2552. Strikingly, the growth defect was partially restored by overexpression of rlmE encoding the SAM-dependent methyltransferase responsible for Um2552. Although SAM is involved not only in rRNA methylation but also in various cellular processes, effects on ribosome biogenesis contribute substantially to the observed defects on cell proliferation.

440 FAS-ligand (FASL) expression in cutaneous T-cell lymphoma (CTCL) is regulated by promoter methylation and can be derepressed by methotrexate (MTX)

One of the major mechanisms of T-cell apoptosis is activation-induced cell death (AICD) - a process by which upregulation of FASL results in apoptosis via the extrinsic (caspase 8-mediated) pathway. Because it is so potentially lethal to T cells, expression of FASL is very tightly controlled. We hypothesized that like many other tumor suppressor genes, FASL expression is at least partially regulated by promoter methylation. Using shRNA technology in-vitro, we show that knockdown of DNA methyltransferase (DNMT) -1 and -3A result in upregulation of FASL mRNA and protein in CTCL lines. We also demonstrate a dose-response relationship between the degree of DNMT knockdown and FASL expression. We showed previously that MTX has the ability to inhibit DNA methylation by depleting cellular S-adenosylmethionine, the main methyl donor for DNMTs. Treatment of CTCL lines with MTX increased FASL expression and further enhanced the FASL upregulation induced by DNMT knockdown. These epigenetic changes were associated with increased apoptosis assessed by propidium iodide/Annexin V flow cytometry as well as activation of caspases. These studies document epigenetic regulation of FASL expression by DNMTs and show that MTX can inhibit the suppressive effects of DNMTs resulting in increased CTCL cell death. We know from our prior studies that MTX also increases expression of FAS (the death receptor for FASL). In aggregate, these findings provide a rationale for including MTX in combination therapies designed to enhance CTCL apoptosis. This could include other drugs (e.g. histone deacetylase inhibitors, retinoids, interferons) or physical modalities (e.g. phototherapy or ionizing radiation).

Metabolic perturbation of epigenome by inhibiting S-adenosylhomocysteine hydrolase elicits senescence through DNA damage response in hepatoma cells.

Cellular senescence is a key physiological barrier against tumor and represents an option for therapeutic intervention. One pivotal intracellular stimulus causing senescence is DNA damage response, while the senescence-associated heterochromatin in cancer limits the strength of the DNA damage response to endogenous genotoxic stress or DNA-damaging agents. Therefore, targeting the maintenance of compacted chromatin in cancer cells represents an optional intervention to improve the therapeutic efficacy in cancer treatment. Given a crosstalk between methionine cycle and histone methylation, we hypothesize that pharmacologically disrupting methylation potential, defined as the ratio of cellular S-adenosylmethionine to S-adenosylhomocysteine, could affect the chromatin structures in cancer cells and thus enhance their sensitivity to DNA damage response signaling. Our results showed that 3-deazaneplanocin A, a chemical inhibitor of S-adenosylhomocysteine hydrolase, elicited a typical cellular senescence in hepatoma cells. Therapy-induced senescence by 3-deazaneplanocin A was mediated through p53-p21 pathway and triggered by enhanced ataxia-telangiectasia mutated activation related to chromatin changes. In conclusion, our study demonstrated that metabolic perturbation of chromatin status in oncogene-activated cancers could be an optional intervention to sensitize DNA damage response signaling.

Membrane Lipids Speak to Histones

In this issue of Molecular Cell, Ye etal. (2017) use a combination of genetic, metabolomic, and transcriptomic approaches to explore the potential for methionine metabolism to influence signal transduction and gene expression in budding yeast.

Characterization and redox regulation of Plasmodium falciparum methionine adenosyltransferase.

As a methyl group donor for biochemical reactions, S-adenosylmethionine plays a central metabolic role in most organisms. Depletion of S-adenosylmethionine has downstream effects on polyamine metabolism and methylation reactions, and is an effective way to combat pathogenic microorganisms such as malaria parasites. Inhibition of both the methylation cycle and polyamine synthesis strongly affects Plasmodium falciparum growth. Despite its central position in the methylation cycle, not much is currently known about P. falciparum methionine adenosyltransferase (PfalMAT). Notably, however, PfalMAT has been discussed as a target of different redox regulatory modifications. Modulating the redox state of critical cysteine residues is a way to regulate enzyme activity in different pathways in response to changes in the cellular redox state. In the present study, we optimized an assay for detailed characterization of enzymatic activity and redox regulation of PfalMAT. While the presence of reduced thioredoxin increases the activity of the enzyme, it was found to be inhibited upon S-glutathionylation and S-nitrosylation. A homology model and site-directed mutagenesis studies revealed a contribution of the residues Cys52, Cys113 and Cys187 to redox regulation of PfalMAT by influencing its structure and activity. This phenomenon connects cellular S-adenosylmethionine synthesis to the redox state of PfalMAT and therefore to the cellular redox homeostasis.

S-adenosylmethionine mediates inhibition of inflammatory response and changes in DNA methylation in human macrophages.

S-adenosylmethionine (SAM), the unique methyl donor in DNA methylation, has been shown to lower lipopolysaccharide (LPS)-induced expression of the proinflammatory cytokine TNF-α and increase the expression of the anti-inflammatory cytokine IL-10 in macrophages. The aim of this study was to assess whether epigenetic mechanisms mediate the anti-inflammatory effects of SAM. Human monocytic THP1 cells were differentiated into macrophages and treated with 0, 500, or 1,000 μmol/l SAM for 24 h, followed by stimulation with LPS. TNFα and IL-10 expression levels were measured by real-time PCR, cellular concentrations of SAM and S-adenosylhomocysteine (SAH), a metabolite of SAM, were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and DNA methylation was measured with LC-MS/MS and microarrays. Relative to control (0 μmol/l SAM), treatment with 500 μmol/l SAM caused a significant decrease in TNF-α expression (-45%, P < 0.05) and increase in IL-10 expression (+77%, P < 0.05). Treatment with 1,000 μmol/l SAM yielded no significant additional benefits. Relative to control, 500 μmol/l SAM increased cellular SAM concentrations twofold without changes in SAH, and 1,000 μmol/l SAM increased cellular SAM sixfold and SAH fourfold. Global DNA methylation increased 7% with 500 μmol/l SAM compared with control. Following treatment with 500 μmol/l SAM, DNA methylation microarray analysis identified 765 differentially methylated regions associated with 918 genes. Pathway analysis of these genes identified a biological network associated with cardiovascular disease, including a subset of genes that were differentially hypomethylated and whose expression levels were altered by SAM. Our data indicate that SAM modulates the expression of inflammatory genes in association with changes in specific gene promoter DNA methylation.

Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer

Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70–75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell.