Methyl Donor S-adenosylmethionine Research Articles

Depletion of S-adenosylmethionine induced by arsenic exposure is involved in liver injury of rat through perturbing histone H3K36 trimethylation dependent bile acid metabolism

Long-term exposure to arsenic, a common environmental pollutant, can induce various types of liver injury, but the mechanism and treatment measures remain unclear. This study constructed a rat model of arsenic-induced liver injury, with methyl group donor S-adenosylmethionine (SAM) supplementation and Rosa roxburghii Tratt juice intervention, to explore the epigenetic mechanism and intervention method of arsenic-induced liver injury from the perspective of hepatic bile acid metabolism. The results showed that arsenic exposure induced the accumulation of total bile acids (TBA) in the liver and serum of rats, and the abnormalities in liver function and liver histopathology. Arsenic reduced histone H3K36 trimethylation (H3K36me3) in the liver via consuming methyl group donor SAM. The reduction of H3K36me3 was involved in arsenic-induced bile acid accumulation by inhibiting the transcription of negative feedback regulators Fxr and Fgfr4 for hepatic bile acid synthesis. SAM supplementation reversed arsenic-induced bile acid accumulation and liver injury by reactivating H3k36me3-dependent transcription of Fxr and Fgfr4. Moreover, this study found that Rosa roxburghii Tratt juice could rescue arsenic-induced SAM consumption, recover H3K36me3-dependent negative feedback regulation of hepatic bile acid synthesis, and alleviate arsenic-induced bile acid accumulation and liver injury. In conclusion, arsenic exposure perturbed H3K36me3-dependent hepatic bile acid metabolism via depleting SAM, thereby inducing hepatic bile acid accumulation and liver injury, which was ameliorated by the supporting effect of Rosa roxburghii Tratt juice on SAM. This study contributes to understanding the mechanism of arsenic-induced liver injury from the perspective of SAM-dependent epigenetics, providing new insight into its prevention and treatment.

S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is incurable because of the inevitable development of drug resistance. Methionine adenosyltransferase 2α (MAT2A) is the primary producer of the methyl donor S-adenosylmethionine (SAM) and several studies have documented MAT2A deregulation in different solid cancers. As the role of MAT2A in MM has not been investigated yet, the aim of this study was to clarify the potential role and underlying molecular mechanisms of MAT2A in MM, exploring new therapeutic options to overcome drug resistance. By analyzing publicly available gene expression profiling data, MAT2A was found to be more highly expressed in patient-derived myeloma cells than in normal bone marrow plasma cells. The expression of MAT2A correlated with an unfavorable prognosis in relapsed patients. MAT2A inhibition in MM cells led to a reduction in intracellular SAM levels, which resulted in impaired cell viability and proliferation, and induction of apoptosis. Further mechanistic investigation demonstrated that MAT2A inhibition inactivated the mTOR-4EBP1 pathway, accompanied by a decrease in protein synthesis. MAT2A targeting in vivo with the small molecule compound FIDAS-5 was able to significantly reduce tumor burden in the 5TGM1 model. Finally, we found that MAT2A inhibition can synergistically enhance the anti-MM effect of the standard-of-care agent bortezomib on both MM cell lines and primary human CD138+ MM cells. In summary, we demonstrate that MAT2A inhibition reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, our findings suggest that the MAT2A inhibitor FIDAS-5 could be a novel compound to improve bortezomib-based treatment of MM.

Broad-Spectrum Small-Molecule Inhibitors Targeting the SAM-Binding Site of Flavivirus NS5 Methyltransferase.

Flavivirus infections, such as those caused by dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV), pose a rising threat to global health. There are no FDA-approved drugs for flaviviruses, although a small number of flaviviruses have vaccines. For flaviviruses or unknown viruses that may appear in the future, it is particularly desirable to identify broad-spectrum inhibitors. The NS5 protein is regarded as one of the most promising flavivirus drug targets because it is conserved across flaviviruses. In this study, we used FL-NAH, a fluorescent analog of the methyl donor S-adenosyl methionine (SAM), to develop a fluorescence polarization (FP)-based high throughput screening (HTS) assay to specifically target methyltransferase (MTase), a vital enzyme for flaviviruses that methylates the N7 and 2'-O positions of the viral 5'-RNA cap. Pilot screening identified two candidate MTase inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the DENV3 MTase with low micromolar IC50. Functional assays verified the inhibitory potency of these molecules for the flavivirus MTase activity. Binding studies indicated that these molecules are bound directly to the DENV3 MTase with similar low micromolar affinity. Furthermore, we showed that these compounds greatly reduced ZIKV replication in cell-based experiments at dosages that did not cause cytotoxicity. Finally, docking studies revealed that these molecules bind to the SAM-binding region on the DENV3 MTase, and further mutagenesis studies verified residues important for the binding of these compounds. Overall, these compounds are innovative and attractive candidates for the development of broad-spectrum inhibitors for the treatment of flavivirus infections.

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.

Sexual differentiation in human malaria parasites is regulated by competition between phospholipid metabolism and histone methylation.

For Plasmodium falciparum, the most widespread and virulent malaria parasite that infects humans, persistence depends on continuous asexual replication in red blood cells, while transmission to their mosquito vector requires asexual blood-stage parasites to differentiate into non-replicating gametocytes. This decision is controlled by stochastic derepression of a heterochromatin-silenced locus encoding AP2-G, the master transcription factor of sexual differentiation. The frequency of ap2-g derepression was shown to be responsive to extracellular phospholipid precursors but the mechanism linking these metabolites to epigenetic regulation of ap2-g was unknown. Through a combination of molecular genetics, metabolomics and chromatin profiling, we show that this response is mediated by metabolic competition for the methyl donor S-adenosylmethionine between histone methyltransferases and phosphoethanolamine methyltransferase, a critical enzyme in the parasite's pathway for de novo phosphatidylcholine synthesis. When phosphatidylcholine precursors are scarce, increased consumption of SAM for de novo phosphatidylcholine synthesis impairs maintenance of the histone methylation responsible for silencing ap2-g, increasing the frequency of derepression and sexual differentiation. This provides a key mechanistic link that explains how LysoPC and choline availability can alter the chromatin status of the ap2-g locus controlling sexual differentiation.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Peripheral T-cell lymphomas (PTCL) are a rare and heterogeneous group of malignancies that share a unifying feature of epigenetic dysregulation. Recent evidence suggests that PTCL, unlike other forms of cancer or even lymphoma, exhibits a marked vulnerability to hypomethylating agents (HMAs) alone and especially in combination with HDAC inhibitors. HMAs like azacitidine (AZA) and decitabine (DAC) have been shown to reverse transcriptional repression secondary to hypermethylation. Cladribine (CLAD) is FDA-approved for the treatment of hairy cell leukemia and is postulated to inhibit both DNA and histone methylation. Here, we sought to define the mechanistic differences between different DNA HMAs in order to inform a rationale on the optimal combinations that might exploit the epigenetic vulnerabilities of PTCL. Methods: Cell viability, caspase activity assay, and western blotting (WB) were performed on a panel of T-cell lymphoma cell lines to identify the effects of AZA/DAC/CLAD on cell survival, apoptosis, and DNA methyltransferase (DNMT) levels. Liquid chromatography-based mass spectrometry (LC-MS) was done to quantify 5-methyl cytosine (5-mC) levels. Results: Cell viability analysis confirmed that the dose and time-dependent sensitivity of the six PTCL cell lines differed following exposure to AZA/DAC/CLAD, where CLAD showed the lowest IC50 value (0.1–1 µM) across all cell lines, followed by AZA (IC50: 1–10 µM), and DAC had the least effect on cell viability (IC50: >100 µM, except T-ALL). A differential threshold of caspase activation was observed across the PTCL cell lines where CLAD showed the most superior caspase activity leading to the highest apoptotic potential. However, WB analysis showed that after 24 hours of treatment, DNMT1 and DNMT3A protein levels were significantly depleted at low concentrations of DAC (<0.01 µM) compared to AZA whereas CLAD did not affect the DNMT1 and DNMT3A levels. LC-MS-based quantification of 5-mC showed a similar relationship in methylation, with initial changes observed at doses >0.01 μM of DAC and >0.1 μM of AZA/CLAD. Taking the short half-life of AZA/DAC into account, cell viability assays with daily addition of AZA/DAC showed increased cell cytotoxicity for AZA/DAC treated samples, which was comparable to CLAD. Ongoing analyses of gene expression and metabolism of the universal methyl donor S-adenosylmethionine (SAM) will help to understand the mechanistic difference between these HMAs. Conclusions: These data suggest that the HMAs have a distinct mechanism of action for hypomethylation. While AZA/DAC induces hypomethylation by depletion of DNMTs, CLAD acts in a DNMT depletion-independent pathway. A mechanistic understanding of AZA/DAC/CLAD will inform how best to use them in clinic as well as assist in the strategic development of biological correlates to further support their therapeutic application. The research was funded by: Translational Orphan Blood Cancer Research Initiative Fund and RO1 # FD-R-006814-01. Keywords: Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Abstract 4744: Epigenetic reprogramming induced by lipids fosters mammary cell plasticity in non-transformed breast epithelial cells

Abstract Introduction. Lipid exposure of non-transformed breast epithelial cells results in increased flux modifications, and profound changes in gene expression. The upregulated genes are involved in stemness, neural development, and neural crest pathways. Neural genes are highly expressed in an estrogen receptor negative breast cancer (ERnegBC): the triple negative subtype. We aim to identify mechanisms that link lipids and epigenetic reprogramming to the genesis of ERnegBC. Methods. Non-transformed MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. S-adenosylmethionine (SAM), 2-HG and other metabolites were analyzed following treatment with OA ± PHGDH inhibitor. CUT&amp;RUN for H3K4me3 and H3K27me3 was performed, and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. OA-induced genes with CUT&amp;RUN peaks were measured in breast organoids derived from reduction mammoplasty samples exposed to OA. The Aldefluour assay was used to identify stem-like (ALDH+) cells. Results. U13C-glucose tracing in presence of OA showed decreased glucose uptake, glycolysis and pentose phosphate pathway flux, with little effect on the TCA cycle. One-carbon (1C)-THF was redirected to the methionine cycle increasing flux to methylation. The methyl donor SAM and the demethylase inhibitor 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases likely via effects on 1C metabolism. H3K4me3 CUT&amp;RUN revealed 661 differential peaks (FDR &amp;lt; 0.05) comparing OA to control. The genes associated with these peaks are involved in neuron differentiation, neural crest, EMT and neural genes expressed in ERnegBC. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR &amp;lt; 0.01), including NGFR (log2FC = 11.7), NGF (log2FC = 8), NTRK1 (log2FC = 2.1). OA-induced genes with enriched H3K4me3 peaks were also upregulated in breast organoids exposed to OA, such as NGFR (FC = 2.2), NGF (FC = 4.7), NTRK1 (FC = 6.6). Motif analysis revealed an overrepresentation of transcription factors (p &amp;lt; 0.05) associated with EMT (Zeb1, Slug), neural functions (E2A, AP1, JunB), neuronal-injury (Atf3) and stress response (Chop, Atf4). Twelve H3K27me3 peaks were enriched in control (FDR &amp;lt; 0.05) and associated with increased gene expression in OA, among them were LGR6 (log2FC = 1.9), a gene associated with ERnegBC and PLAG1 (log2FC = 2.8) a stem cell marker. ALDH+ cells’ percentage increased by at least 10% after OA exposure. Conclusions. Lipid exposure affects the production of SAM and 2-HG, which results in epigenetic fostered plasticity, leading to reprogramming/selecting cells with a multi-potential embryonic or stem-like state and/or differentiation to a neural/neural crest-like state which may facilitate ERnegBC genesis. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Seema Khan, Susan Clare. Epigenetic reprogramming induced by lipids fosters mammary cell plasticity in non-transformed breast epithelial cells. [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 4744.

Abstract 503: ISM3412, a novel and selective MAT2A inhibitor for the treatment of cancer

Abstract The enzyme methionine adenosyltransferase 2α (MAT2A) has an important role in metabolism and epigenetics as the major producer of the universal methyl donor S-adenosyl methionine (SAM). Despite broad cellular functions, MAT2A was recently implicated as a synthetic lethal target in cancers that have a deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is found in approximately 15% of all cancers. Here, we report ISM3412 is a novel potent MAT2A inhibitor with high selectivity. In enzymatic assays, ISM3412 demonstrated a strong inhibition of MAT2A with an IC50 of 19.4 nM. In both HCT116 MTAP-/- cells and HCT116 parental cells, the compound inhibited SAM production, with IC50 values of 5.97 nM and 3.66 nM, respectively. Additionally, it inhibited SDMA production in HCT116 MTAP-/- cells (IC50 of 6.37 nM), but not in HCT116 parental cells (IC50 &amp;gt; 2000 nM). Consistent with the large SDMA inhibitory potency difference between the HCT116 isogenic cell pair, ISM3412 also exhibited very good selectivity in cellular proliferation assays. While it inhibited cellular growth in HCT116 MTAP-/-cells with an IC50 of 199nM, its inhibitory potency on HCT116 parental cells was very weak (IC50 of &amp;gt;21117nM), revealing more than a 100-fold selectivity in inhibiting growth in this isogenic pair cell line. Furthermore, while ISM3412 strongly inhibited proliferation in multiple cancer cell lines with natural MTAP deficiency (including pancreatic cancer, bladder cancer, and NSCLC), the same was not observed in multiple MTAP-intact cell lines (IC50 &amp;gt; 30 uM). Consistent with the in vitro data, ISM3412 demonstrated robust mono-therapeutic efficacy in an MTAP-/- HCT116 CDX model with tumor growth inhibition of 66% at 3 mg/kg QD. Similarly, ISM3412 also demonstrated robust single-agent efficacy in CDX models of pancreatic cancer, bladder cancer and NSCLC, as well as in a PDX model of esophageal cancer. Notably, ISM3412 showed improved efficacy when combined with docetaxel, compared to the individual monotherapy. ISM3412, at 10 uM, showed no obvious agonistic or antagonistic effect on 44 selected targets in a mini safety panel, demonstrating its selectivity as a MAT2A inhibitor. In addition to its strong biological potency and selectivity, ISM3412 exhibited favorable drug-likeness properties, including low molecular weight, good solubility and permeability, and low plasma protein binding. In vivo PK data revealed low clearance and high oral-bioavailability. Further, it was well tolerated with no significant hepatobiliary toxicity. In conclusion, ISM3412 is a novel and highly selective MAT2A inhibitor for MTAP-deficient cancers, and merits clinical evaluation. Citation Format: Qingyuan Meng, Xiao Ding, Xiaosong Liu, Hailong Wang, Ling Wang, Junwen Qiao, Hua Cao, Yushu Yin, Congcong Zhu, Xing Liang, Xin Cai, Yuanyuan Xu, Chenxi Xu, Shan Chen, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM3412, a novel and selective MAT2A inhibitor for the treatment 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 503.

The Lysophospholipase PNPLA7 Controls Hepatic Choline and Methionine Metabolism.

The in vivo roles of lysophospholipase, which cleaves a fatty acyl ester of lysophospholipid, remained unclear. Recently, we have unraveled a previously unrecognized physiological role of the lysophospholipase PNPLA7, a member of the Ca2+-independent phospholipase A2 (iPLA2) family, as a key regulator of the production of glycerophosphocholine (GPC), a precursor of endogenous choline, whose methyl groups are preferentially fluxed into the methionine cycle in the liver. PNPLA7 deficiency in mice markedly decreases hepatic GPC, choline, and several metabolites related to choline/methionine metabolism, leading to various symptoms reminiscent of methionine shortage. Overall metabolic alterations in the liver of Pnpla7-null mice in vivo largely recapitulate those in methionine-deprived hepatocytes in vitro. Reduction of the methyl donor S-adenosylmethionine (SAM) after methionine deprivation decreases the methylation of the PNPLA7 gene promoter, relieves PNPLA7 expression, and thereby increases GPC and choline levels, likely as a compensatory adaptation. In line with the view that SAM prevents the development of liver cancer, the expression of PNPLA7, as well as several enzymes in the choline/methionine metabolism, is reduced in human hepatocellular carcinoma. These findings uncover an unexplored role of a lysophospholipase in hepatic phospholipid catabolism coupled with choline/methionine metabolism.

S-adenosylmethionine synthases specify distinct H3K4me3 populations and gene expression patterns during heat stress.

Methylation is a widely occurring modification that requires the methyl donor S-adenosylmethionine (SAM) and acts in regulation of gene expression and other processes. SAM is synthesized from methionine, which is imported or generated through the 1-carbon cycle (1 CC). Alterations in 1 CC function have clear effects on lifespan and stress responses, but the wide distribution of this modification has made identification of specific mechanistic links difficult. Exploiting a dynamic stress-induced transcription model, we find that two SAM synthases in Caenorhabditis elegans, SAMS-1 and SAMS-4, contribute differently to modification of H3K4me3, gene expression and survival. We find that sams-4 enhances H3K4me3 in heat shocked animals lacking sams-1, however, sams-1 cannot compensate for sams-4, which is required to survive heat stress. This suggests that the regulatory functions of SAM depend on its enzymatic source and that provisioning of SAM may be an important regulatory step linking 1 CC function to phenotypes in aging and stress.

Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups.

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

Modulation of DNA methylation by one-carbon metabolism: a milestone for healthy aging.

Healthy aging can be defined as an extended lifespan and health span. Nutrition has been regarded as an important factor in healthy aging, because nutrients, bioactive food components, and diets have demonstrated beneficial effects on aging hallmarks such as oxidative stress, mitochondrial function, apoptosis and autophagy, genomic stability, and immune function. Nutrition also plays a role in epigenetic regulation of gene expression, and DNA methylation is the most extensively investigated epigenetic phenomenon in aging. Interestingly, age-associated DNA methylation can be modulated by one-carbon metabolism or inhibition of DNA methyltransferases. One-carbon metabolism ultimately controls the balance between the universal methyl donor S-adenosylmethionine and the methyltransferase inhibitor S-adenosylhomocysteine. Water-soluble B-vitamins such as folate, vitamin B6, and vitamin B12 serve as coenzymes for multiple steps in one-carbon metabolism, whereas methionine, choline, betaine, and serine act as methyl donors. Thus, these one-carbon nutrients can modify age-associated DNA methylation and subsequently alter the age-associated physiologic and pathologic processes. We cannot elude aging per se but we may at least change age-associated DNA methylation, which could mitigate age-associated diseases and disorders.

The Role of the m6A RNA Methyltransferase METTL16 in Gene Expression and SAM Homeostasis.

The RNA methylation of adenosine at the N6-position (m6A) has attracted significant attention because of its abundance and dynamic nature. It accounts for more than 80% of all RNA modifications present in bacteria and eukaryotes and regulates crucial aspects of RNA biology and gene expression in numerous biological processes. The majority of m6A found in mammals is deposited by a multicomponent complex formed between methyltransferase-like (METTL) proteins METTL3 and METTL14. In the last few years, the list of m6A writers has grown, resulting in an expansion of our understanding of the importance of m6A and the methylation machinery. The characterization of the less familiar family member METTL16 has uncovered a new function of the m6A methylation apparatus, namely the fine-tuning of the cellular levels of the major methyl donor S-adenosylmethionine (SAM). METTL16 achieves this by adjusting the levels of the enzyme that synthesizes SAM in direct response to fluctuations in the SAM availability. This review summarizes recent progress made in understanding how METTL16 can sense and relay metabolic information and considers the wider implications. A brief survey highlights similarities and differences between METTL16 and the better-known METTL3/14 complex, followed by a discussion of the target specificity, modes of action and potential roles of METTL16.

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.

Loss of LEUCINE CARBOXYL METHYLTRANSFERASE 1 interferes with metal homeostasis in Arabidopsis and enhances susceptibility to environmental stresses

The biochemical function of LEUCINE CARBOXYL METHYLTRANSFERASE 1 (LCMT1) is to transfer a methyl group from the methyl donor S-adenosylmethionine (SAM) to the catalytic subunits of PROTEIN PHOSPHATASE 2A (PP2Ac), PP4 and PP6. This post-translational modification by LCMT1 is found throughout eukaryotes from yeast to animals and plants, indicating that its function is essential. However, Arabidopsis with knocked out LCMT1 still grows and develops almost normally, at least under optimal growth conditions. We therefore proposed that the presence of LCMT1 would be important under non-optimal growth conditions and favoured plant survival during evolution. To shed light on the physiological functions of plant LCMT1, phenotypes of the lcmt1 mutant and wild type Arabidopsis were compared under various conditions including exposure to heavy metals, variable chelator concentrations, and increased temperature. The lcmt1 mutant was found to be more susceptible to these environmental changes than wild type and resulted in poor growth of seedlings and rosette stage plants. Element analysis of rosette stage plants mainly showed a difference between the lcmt1 mutant and wild type regarding concentrations of sodium and boron, two-fold up or halved, respectively. In both lcmt1 and wild type, lack of EDTA in the growth medium resulted in enhanced concentration of copper, manganese, zinc and sulphur, and especially lcmt1 growth was hampered by these conditions. The altered phenotype in response to stress, the element and mRNA transcript analysis substantiate that LCMT1 has an important role in metal homeostasis and show that functional LCMT1 is necessary to prevent damages from heat, heavy metals or lack of chelator.

NNMT promotes the progression of intrahepatic cholangiocarcinoma by regulating aerobic glycolysis via the EGFR-STAT3 axis

Nicotinamide N-methyltransferase (NNMT), a member of the N-methyltransferase family, plays an important role in tumorigenesis. However, its expression and biological functions in intrahepatic cholangiocarcinoma (iCCA) remain to be established. In our study, we identified NNMT as an oncogene in iCCA and provided mechanistic insights into the roles of NNMT in iCCA progression. High NNMT expression in iCCA tissues was identified using western blotting and immunohistochemistry (IHC). We identified a significantly higher NNMT expression level in human iCCA tissues than that in adjacent normal tissues. Increased NNMT expression promoted iCCA cell proliferation and metastasis in vitro and in vivo. Mechanistically, NNMT inhibited the level of histone methylation in iCCA cells by consuming the methyl donor S-adenosyl methionine (SAM), thereby promoting the expression of epidermal growth factor receptor (EGFR). EGFR may activate the aerobic glycolysis pathway in iCCA cells by activating the STAT3 signaling pathway. In conclusion, we identified NNMT as an oncogene in iCCA and provided mechanistic insights into the roles of NNMT in iCCA progression.

Plasmodium falciparum S-Adenosylmethionine Synthetase Is Essential for Parasite Survival through a Complex Interaction Network with Cytoplasmic and Nuclear Proteins.

S-adenosylmethionine synthetase (SAMS) is a key enzyme for the synthesis of the lone methyl donor S-adenosyl methionine (SAM), which is involved in transmethylation reactions and hence required for cellular processes such as DNA, RNA, and histone methylation, but also polyamine biosynthesis and proteostasis. In the human malaria parasite Plasmodium falciparum, PfSAMS is encoded by a single gene and has been suggested to be crucial for malaria pathogenesis and transmission; however, to date, PfSAMS has not been fully characterized. To gain deeper insight into the function of PfSAMS, we generated a conditional gene knockdown (KD) using the glmS ribozyme system. We show that PfSAMS localizes to the cytoplasm and the nucleus of blood-stage parasites. PfSAMS-KD results in reduced histone methylation and leads to impaired intraerythrocytic growth and gametocyte development. To further determine the interaction network of PfSAMS, we performed a proximity-dependent biotin identification analysis. We identified a complex network of 1114 proteins involved in biological processes such as cell cycle control and DNA replication, or transcription, but also in phosphatidylcholine and polyamine biosynthesis and proteasome regulation. Our findings highlight the diverse roles of PfSAMS during intraerythrocytic growth and sexual stage development and emphasize that PfSAMS is a potential drug target.

Homocysteine Metabolism in Pregnancy and Developmental Impacts.

Homocysteine is a metabolite generated by methionine cycle metabolism, comprising the demethylated derivative of methionine. Homocysteine can be metabolised by the transsulphuration pathway to cystathionine, which requires vitamin B6, or can undergo remethylation to methionine. Homocysteine remethylation to methionine is catalysed by methionine synthase activity which requires vitamin B12, regenerating methionine to allow synthesis of the universal methyl donor S-adenosylmethionine required for methylation and gene transcription regulation. The methyl-group donated for homocysteine remethylation comes from 5-methyltetrahydrofolate generated by the folate cycle, which allows tetrahydrofolate to be returned to the active folate pool for nucleotide biosynthesis. Therefore the integrated actions of the methionine and folate cycles, required to metabolise homocysteine, also perpetuate methylation and nucleotide synthesis, vitally important to support embryonic growth, proliferation and development. Dysregulated activities of these two interdependent metabolic cycles, arising from maternal suboptimal intake of nutrient co-factors such as folate and vitamin B12 or gene polymorphisms resulting in reduced enzymatic activity, leads to inefficient homocysteine metabolic conversion causing elevated concentrations, known as hyperhomocysteinemia. This condition is associated with multiple adverse pregnancy outcomes including neural tube defects (NTDs). Raised homocysteine is damaging to cellular function, binding to proteins thereby impairing their function, with perturbed homocysteine metabolism impacting negatively on embryonic development. This review discusses the “cross-talk” of maternal-fetal homocysteine interrelationships, describes the placental transport of homocysteine, homocysteine impacts on pregnancy outcomes, homocysteine and methylation effects linking to NTD risk and proposes a putative pathway for embryonic provision of folate and vitamin B12, homocysteine-modulating nutrients that ameliorate NTD risk.

Abstract 3745: Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression

Abstract Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer types, including colorectal cancer. Abnormality of several factors, such as DNA methyltransferases (DNMT), demethylases, or deviation in methyl-donor (folate and S-adenosylmethionine) availability can contribute to the development of genome-wide hypomethylation. Detection of epigenetic changes as global DNA hypomethylation in cell-free DNA fraction obtained from blood samples can expand the opportunities for the early recognition of colorectal cancer. One of our main goals was the investigation of global DNA methylation patterns in tissue biopsies (n=183) and cell-free DNA fraction of blood samples (n=48) along the colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore possible underlying mechanisms of genome-wide hypomethylation formation in 12 colorectal tumor tissue sections, containing transition zones. Using LINE-1 pyrosequencing, significantly reduced global DNA methylation level was detected in line with cancer progression in tissue specimens (normal: 77.5±1.7%, adenoma: 72.7±4.8%, carcinoma: 69.7±7.6%, p≤0.0001) and in liquid biopsies as well (normal: 82.0±2.0%, adenoma: 80.0±1.7%, carcinoma: 79.8±1.3%, p≤0.01). However, no significant methylation changes were found in inflammatory bowel disease cases. Analyzing microarray data in silico, altered mRNA expression of certain methylation-, and one-carbon metabolism-related genes were detected in tumorous specimens vs. healthy biopsies, from which DNMT1 was upregulated, and folate receptor 2 (FOLR2) was downregulated. DNMT and FOLR2 expression were validated by immunohistochemistry. Furthermore, significantly reduced folic acid and S-adenosylmethionine content were observed in parallel with diminishing 5-methylcytosine levels in adenoma and carcinoma sections compared to normal adjacent to tumor tissue areas by immunolabeling (p≤0.05). Our results suggest that intraindividual monitoring of genome-wide hypomethylation may assist in the recognition of adenoma formation, cancer progression, or remission as well. Moreover, lower global DNA methylation level could be connected to decreased methyl-donor availability with the contribution of reduced FOLR2 expression. Citation Format: Krisztina Andrea Szigeti, Alexandra Kalmár, Gábor Valcz, Barbara Kinga Barták, Zsófia Nagy, Sára Zsigrai, Ildikó Felletár, Árpád V Patai, Tamás Micsik, Márton Papp, Eszter Márkus, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3745.

Epigenetic Modifications and Their Potential Contribution to Traumatic Brain Injury Pathobiology and Outcome.

Epigenetic information is not permanently encoded in the DNA sequence, but rather consists of reversible, heritable modifications that regulate the gene expression profile of a cell. Epigenetic modifications can result in cellular changes that can be long lasting and include DNA methylation, histone methylation, histone acetylation, and RNA methylation. As epigenetic modifications are reversible, the enzymes that add (epigenetic writers), the proteins that decode (epigenetic readers), and the enzymes that remove (epigenetic erasers) these modifications can be targeted to alter cellular function and disease biology. While epigenetic modifications and their contributions are intense topics of current research in the context of a number of diseases, including cancer, inflammatory diseases, and Alzheimer disease, the study of epigenetics in the context of traumatic brain injury (TBI) is in its infancy. In this review, we will summarize the experimental and clinical findings demonstrating that TBI triggers epigenetic modifications, with a focus on changes in DNA methylation, histone methylation, and the translational utility of the universal methyl donor S-adenosylmethionine (SAM). Finally, we will review the evidence for using methyl donors as possible treatments for TBI-associated pathology and outcome.