Methyl Donor S-adenosylmethionine Research Articles

S-Adenosylmethionine affects ERK1/2 and STAT3 pathway in androgen-independent prostate cancer cells

BackgroundThe most critical point in the treatment of prostate cancer is the progression towards a hormone-refractory tumour, making research on alternative therapies necessary. This study focused on the methyl donor S-adenosylmethionine (SAM), which is known to act as an antitumourigenic in several cancer cell lines. Though a genome-wide downregulation of proto-oncogenes in prostate cancer cell lines treated with SAM is obvious, the anticancer effects remain elusive. Thus, in this study, the impact of SAM treatment on the cell cycle, apoptosis and cancer-related pathways was investigated.Methods and resultsAfter performing SAM treatment on prostate cancer cell lines (PC-3 and DU145), a cell-cycle arrest during the S-phase, a downregulation of cyclin A protein levels and an upregulation of p21 cell cycle inhibitor were observed. The proapoptotic Bax/Bcl-2 ratio and the caspase-3 activity were elevated; additionally, the apoptosis rate of SAM treated cells increased significantly in a time-dependent manner. Moreover, immunoblots displayed a downregulation of Erk1/2 and STAT3 phosphorylation accompanied by a reduced expression of the STAT3 protein.ConclusionSAM caused changes in cancer-related pathways, probably leading to the effects on the cell cycle and apoptosis rate. These results provide deeper insights into the anticancer effects of SAM on prostate cancer cells.

Heritable shifts in redox metabolites during mitochondrial quiescence reprogramme progeny metabolism.

Changes in maternal diet and metabolic defects in mothers can profoundly impact progeny health and disease. However, the biochemical mechanisms that induce the initial reprogramming events at the cellular level have remained largely unknown due to limitations in obtaining pure populations of quiescent oocytes. Here, we show that the precocious onset of Mitochondrial Respiratory Quiescence (MRQ) causes a reprogramming of progeny metabolic state. The premature onset of MRQ drives the lowering of Drosophila oocyte NAD+ levels. NAD+ depletion in the oocyte leads to reduced methionine cycle production of the methyl donor S-adenosylmethionine (SAM) in embryos and lower H3K27-me3 levels, resulting in enhanced levels of progeny intestinal lipid metabolism. In addition, we show that triggering cellular quiescence in mammalian cells and chemotherapy-resistant human cancer cell models induces cellular reprogramming events identical to those seen in Drosophila, suggesting a conserved metabolic mechanism in systems reliant on quiescent cells.

Role of Epigenetic Therapy in the Modulation of Tumor Growth and Migration in Human Castration-Resistant Prostate Cancer Cells with Neuroendocrine Differentiation

Introduction: Neuroendocrine transdifferentiation (NED) of prostate cancer (PC) cells is associated with the development of resistance to antiandrogen therapy and poor prognosis in patients with castration-resistant PC (CRPC). Many of the molecular events, involved in NED, appear to be mediated by epigenetic mechanisms. In this study, we evaluated the antitumor activity and epigenetic modulation of 2 epigenetic drugs, such as the demethylating agent 5-aza-2′-deoxycytidine (AZA) and the methyl donor S-adenosylmethionine (SAM), in 2 human CRPC cell lines with NED (DU-145 and PC-3). Methods: The effects of AZA and SAM on cell viability, cell cycle, apoptosis, migration, and genome-wide DNA methylation profiling have been evaluated. Results: Both drugs showed a prominent antitumor activity in DU-145 and PC-3 cells, through perturbation of cell cycle progression, induction of apoptosis, and inhibition of cell migration. AZA and SAM reversed NED in DU-145 and PC-3, respectively. Moreover, AZA treatment modified DNA methylation pattern in DU-145 cells, sustaining a pervasive hypomethylation of the genome, with a relevant effect on several pathways involved in the regulation of cell proliferation, apoptosis, and cell migration, in particular Wnt/β-catenin. Conclusions: A relevant antitumor activity of these epigenetic drugs on CRPC cell lines with NED opens a new scenario in the therapy of this lethal variant of PC.

Reduction of DNMT3a and RORA in the nucleus accumbens plays a causal role in post-traumatic stress disorder-like behavior: reversal by combinatorial epigenetic therapy.

Post-traumatic stress disorder (PTSD) is an incapacitating trauma-related disorder, with no reliable therapy. Although PTSD has been associated with epigenetic alterations in peripheral white blood cells, it is unknown where such changes occur in the brain, and whether they play a causal role in PTSD. Using an animal PTSD model, we show distinct DNA methylation profiles of PTSD susceptibility in the nucleus accumbens (NAc). Data analysis revealed overall hypomethylation of different genomic CG sites in susceptible animals. This was correlated with the reduction in expression levels of the DNA methyltransferase, DNMT3a. Since epigenetic changes in diseases involve different gene pathways, rather than single candidate genes, we next searched for pathways that may be involved in PTSD. Analysis of differentially methylated sites identified enrichment in the RAR activation and LXR/RXR activation pathways that regulate Retinoic Acid Receptor (RAR) Related Orphan Receptor A (RORA) activation. Intra-NAc injection of a lentiviral vector expressing either RORA or DNMT3a reversed PTSD-like behaviors while knockdown of RORA and DNMT3a increased PTSD-like behaviors. To translate our results into a potential pharmacological therapeutic strategy, we tested the effect of systemic treatment with the global methyl donor S-adenosyl methionine (SAM), for supplementing DNA methylation, or retinoic acid, for activating RORA downstream pathways. We found that combined treatment with the methyl donor SAM and retinoic acid reversed PTSD-like behaviors. Thus, our data point to a novel approach to the treatment of PTSD, which is potentially translatable to humans.

Abstract 1025: Genomic and metabolomic analysis of MAT2A inhibition reveals increased RNA splicing, lipid metabolism and cell cycle arrest in MTAP deleted tumor models

Abstract Background: Approximately 15% of solid tumors harbor deletions in Methylthioadenosine phosphorylase (MTAP) (1). MTAP deletion confers enhanced sensitivity to Methionine Adenosyltransferase 2A (MAT2A) inhibition, which synthesizes the universal methyl donor and PRMT5 substrate S-Adenosyl Methionine (SAM) (2). MTAP loss results in the accumulation of its substrate methylthioadenosine (MTA) which partially inhibits PRMT5. PRMT5 symmetrically di-methylates arginine (SDMA) residues on proteins that are key to genomic integrity and proteostasis. Therefore, MTA accumulation and MAT2A inhibitor-driven abrogation of SAM significantly inhibit PRMT5 activity and key oncogenic signaling in MTAP deleted cancers. In this study, we sought to investigate the mechanistic and phenotypic implications of this synthetic lethal relationship. Methods: Using the HCT116 MTAP deleted isogenic pair and a panel of MTAP deleted cell lines, we evaluated the genomic and metabolomic effects of a proprietary MAT2A inhibitor using RNA sequencing (RNA-seq) and untargeted metabolomics respectively. Further, we investigated the effects of MAT2A inhibition across a large panel of MTAP WT and deleted cell lines in a multiplexed assay that assessed proliferation, apoptosis and cell cycle status. Results: MAT2A inhibition decreased SAM, and selectively decreased the proliferation and expression of SDMA in MTAP deleted cell lines. From RNA-seq data, MAT2A inhibition notably affected genes involved in p53 and plasma membrane signaling in the HCT116 MTAP deleted cell line compared to its parental counterpart. RNA-seq data also identified elevated frequency of alternative spliced transcripts that are involved in cell cycle, RNA processing and lipid metabolism pathways. In alignment with these observations, untargeted metabolomic analysis of MAT2A inhibition identified robust and selective changes in polyamine synthesis, oxidative stress and lipid metabolism in the HCT116 MTAP deleted cell line. With the changes in cell cycle related genes and previous reports indicating a role of lipid metabolism in cell cycle arrest , MAT2A inhibition increased expression of the G2/M cell cycle marker phospho-histone H3 and decreased proliferation in a larger percentage of MTAP deleted cell lines compared to MTAP WT cell lines. We further confirmed RNA-Seq data and observed increased p53 expression and a concordant decrease in the p53-regulated G2/M transition marker Cyclin B1 upon MAT2A inhibition in the HCT116 MTAP-deleted cell line. Conclusion: We have shown that MAT2A blockade results in distinct genomic and metabolomic effects that drives G2/M cell cycle arrest in MTAP deleted cell lines. Further analysis of these changes will help identify potential biomarkers that can further inform on the activity of MAT2A inhibition. We are currently targeting to enter clinical trials in 2021. Citation Format: Neil Bhola, Atieh Givmanesh, John Faulhaber, Melissa Fleury, Mark Lackner, Zineb Mounir, Divya Pankajakshan. Genomic and metabolomic analysis of MAT2A inhibition reveals increased RNA splicing, lipid metabolism and cell cycle arrest in MTAP deleted tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1025.

Abstract 1278: MAT2A inhibitor, IDE397, displays broad anti-tumor activity across a panel of MTAP-deleted patient-derived xenografts

Abstract Background: Approximately 15% of all solid tumors harbor deletions in methylthioadenosine phosphorylase (MTAP) (1). MTAP deficiencies have been reported to sensitize tumor cells to methionine adenosyltransferase 2A (MAT2A) modulation (2). MAT2A synthesizes the universal methyl donor and PRMT5 substrate S-adenosylmethionine (SAM) from methionine. PRMT5 symmetrically di-methylates arginine (SDMA) residues on proteins that are key to genomic integrity and proteostasis. We evaluated the anti-tumor activity of the small molecule MAT2A inhibitor IDE397 in multiple CDX models and in an ongoing study of 45 MTAP-deleted PDX models to inform clinical strategy for a First-in-Human clinical trial evaluating IDE397. Methods: CDX models utilized included the HCT116 isogenic pair and endogenous MTAP-deleted models. A series of dose response and time-course studies were performed to inform the efficacy-PD relationship. SAM was quantified by LC-MS and SDMA was visualized by western blot (WB) and further quantified by immunohistochemistry (IHC). PDX models with MTAP homozygous deletions were selected from bladder, esophageal, gastric, head and neck, NSCLC and pancreatic cancers. MTAP deletion was determined by WES and/or SNP 6.0 microarray and verified by absence of significant mRNA. Tumors were subcutaneously implanted into immunocompromised mice by serial transplantation of tumor fragments. Each PDX model included a vehicle control and an IDE397-treated group (n=5/group). IDE397 was administered orally once per day (QD). Tumor growth inhibition (TGI) was calculated using the formula TGI = (TV controlfinal - TV treatedfinal)/(TV controlfinal - TV controlinitial) x 100. Results: Administration of IDE397 resulted in TGI and/or tumor regression in MTAP-deleted CDX and PDX models. The HCT116 MTAP-deleted CDX model was more sensitive to IDE397 compared to the HCT116 MTAP-WT model. IDE397 produced a dose- and time-dependent modulation of the proximal and distal PD biomarkers SAM and SDMA. SAM was decreased in plasma and tumors irrespective of MTAP status. In contrast, SDMA was selectively modulated in MTAP-null models only. In a NSCLC CDX model, a dose dependent TGI was observed, with the higher doses leading to tumor regression. Anti-tumor activity is observed in MTAP-deleted PDX models, where IDE397 administration has resulted in TGI and tumor regressions. Conclusion: Xenograft studies indicate that IDE397 exhibits anti-tumor activity as a single agent in MTAP-deleted CDX models and in MTAP-deleted PDX models of NSCLC, pancreatic, bladder, head and neck, esophageal and gastric cancer. This data supports the clinical evaluation of IDE397 across a wide range of solid tumor types with MTAP-deletion.

The methyl donor S-adenosyl methionine reverses the DNA methylation signature of chronic neuropathic pain in mouse frontal cortex.

To determine the effect of long-term therapeutic exposure to SAM on the DNA methylation of individual genes and pathways in a mouse neuropathic pain model. Male CD-1 mice received spared nerve injury or sham surgery. Three months after injury, animals received SAM (20 mg/kg, oral, 3× a week) or vehicle for 16 weeks followed by epigenome-wide analysis of frontal cortex. Peripheral neuropathic pain was associated with 4000 differentially methylated genomic regions that were enriched in intracellular signaling, cell motility and migration, cytoskeletal structure, and cell adhesion pathways. A third of these differentially methylated regions were reversed by SAM treatment (1415 regions representing 1013 genes). More than 100 genes with known pain-related function were differentially methylated after nerve injury; 29 of these were reversed by SAM treatment including Scn10a, Trpa1, Ntrk1, and Gfap. These results suggest a role for the epigenome in the maintenance of chronic pain and advance epigenetic modulators such as SAM as a novel approach to treat chronic pain.

Depletion of S-adenosylmethionine pool and promoter hypermethylation of Arsenite methyltransferase in arsenic-induced skin lesion individuals: A case-control study from West Bengal, India

Methylation of arsenic compounds in the human body occurs following a series of biochemical reactions in the presence of methyl donor S-adenosylmethionine (SAM) and catalyzed by arsenite methyltransferase (AS3MT). However, the extent and pattern of methylation differs among the arsenic exposed individuals leading to differential susceptibility. The mechanism for such inter-individual difference is enigmatic. In the present case-control study we recruited exposed individuals with and without arsenic induced skin lesion (WSL and WOSL), and an unexposed cohort, each having 120 individuals. Using ELISA, we observed a reduction in SAM levels (p < 0.05) in WSL compared to WOSL. Linear regression analysis revealed a negative correlation between urinary arsenic concentration and SAM concentration between the study groups. qRT-PCR revealed a significant down-regulation (p < 0.01) of key regulatory genes like MTHFR, MTR, MAT2A and MAT2B of SAM biogenesis pathway in WSL cohort. Methylation-specific PCR revealed significant promoter hypermethylation of AS3MT (WSL vs. WOSL: p < 0.01) which resulted in its subsequent transcriptional repression (WSL vs. WOSL: p < 0.001). Linear regression analysis also showed a negative correlation between SAM concentration and percentage of promoter methylation. Taken together, these results indicate that reduction in SAM biogenesis along with a higher utilization of SAM results in a decreased availability of methyl donor. These along with epigenetic down-regulation of AS3MT may be responsible for higher susceptibility in arsenic exposed individuals.

Polycystic ovary syndrome is transmitted via a transgenerational epigenetic process

SummaryPolycystic ovary syndrome (PCOS) is the most common reproductive and metabolic disorder affecting women of reproductive age. PCOS has a strong heritable component, but its pathogenesis has been unclear. Here, we performed RNA sequencing and genome-wide DNA methylation profiling of ovarian tissue from control and third-generation PCOS-like mice. We found that DNA hypomethylation regulates key genes associated with PCOS and that several of the differentially methylated genes are also altered in blood samples from women with PCOS compared with healthy controls. Based on this insight, we treated the PCOS mouse model with the methyl group donor S-adenosylmethionine and found that it corrected their transcriptomic, neuroendocrine, and metabolic defects. These findings show that the transmission of PCOS traits to future generations occurs via an altered landscape of DNA methylation and propose methylome markers as a possible diagnostic landmark for the condition, while also identifying potential candidates for epigenetic-based therapy.

Lipid remodeling in response to methionine stress in MDA-MBA-468 triple-negative breast cancer cells

Methionine (Met) is an essential amino acid and critical precursor to the cellular methyl donor S-adenosylmethionine. Unlike nontransformed cells, cancer cells have a unique metabolic requirement for Met and are unable to proliferate in growth media where Met is replaced with its metabolic precursor, homocysteine. This metabolic vulnerability is common among cancer cells regardless of tissue origin and is known as “methionine dependence”, “methionine stress sensitivity”, or the Hoffman effect. The response of lipids to Met stress, however, is not well-understood. Using mass spectroscopy, label-free vibrational microscopy, and next-generation sequencing, we characterize the response of lipids to Met stress in the triple-negative breast cancer cell line MDA-MB-468 and its Met stress insensitive derivative, MDA-MB-468res-R8. Lipidome analysis identified an immediate, global decrease in lipid abundances with the exception of triglycerides and an increase in lipid droplets in response to Met stress specifically in MDA-MB-468 cells. Furthermore, specific gene expression changes were observed as a secondary response to Met stress in MDA-MB-468, resulting in a downregulation of fatty acid metabolic genes and an upregulation of genes in the unfolded protein response pathway. We conclude that the extensive changes in lipid abundance during Met stress is a direct consequence of the modified metabolic profile previously described in Met stress–sensitive cells. The changes in lipid abundance likely results in changes in membrane composition inducing the unfolded protein response we observe.

The toxic side of one-carbon metabolism and epigenetics

One-carbon metabolism is a central metabolic hub that provides one-carbon units for essential biosynthetic reactions and for writing epigenetics marks. The leading role in this hub is performed by the one-carbon carrier tetrahydrofolate (THF), which accepts formaldehyde usually from serine generating one-carbon THF intermediates in a set of reactions known as the folate or one-carbon cycle. THF derivatives can feed one-carbon units into purine and thymidine synthesis, and into the methionine cycle that produces the universal methyl-donor S-adenosylmethionine (AdoMet). AdoMet delivers methyl groups for epigenetic methylations and it is metabolized to homocysteine (Hcy), which can enter the transsulfuration pathway for the production of cysteine and lastly glutathione (GSH), the main cellular antioxidant. This vital role of THF comes to an expense. THF and other folate derivatives are susceptible to oxidative breakdown releasing formaldehyde, which can damage DNA -a consequence prevented by the Fanconi Anaemia DNA repair pathway. Epigenetic demethylations catalysed by lysine-specific demethylases (LSD) and Jumonji histone demethylases can also release formaldehyde, constituting a potential threat for genome integrity. In mammals, the toxicity of formaldehyde is limited by a metabolic route centred on the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which oxidizes formaldehyde conjugated to GSH, lastly generating formate. Remarkably, this formate can be a significant source of one-carbon units, thus defining a formaldehyde cycle that likely restricts the toxicity of one-carbon metabolism and epigenetic demethylations. This work describes recent advances in one-carbon metabolism and epigenetics, focusing on the steps that involve formaldehyde flux and that might lead to cytotoxicity affecting human health.

Mi-RNA-888-5p Is Involved in S-Adenosylmethionine Antitumor Effects in Laryngeal Squamous Cancer Cells.

Simple SummaryLaryngeal Squamous Cell Carcinoma (LSCC) is a leading cause of cancer-related death with a strong interest in identifying and developing new treatments. MicroRNAs (miRNAs) have emerged as one of the most important determinants of neoplastic transformation and progression. miRNA modulation causes significant antitumor effects both in vitro and in vivo and miRNA regulation by natural compounds, represents a promising approach in the field of cancer research. S-Adenosylmethionine (AdoMet), a natural compound and a nutritional supplement, is well known for its antiproliferative and pro-apoptotic effects in many kinds of human tumors. Here, we report that AdoMet induces ER-stress and autophagy paralleled by miR-888-5p downregulation and MYCBP and CDH1 increased expression in Laryngeal Squamous Cancer Cells (LSCC). This study contributes to understanding the mechanisms by which AdoMet exerts its effects in LSCC, suggesting the use of AdoMet as an attractive miRNA-mediated chemopreventive and therapeutic strategy against cancer.(1) Purpose: The methyl donor S-Adenosylmethionine (AdoMet) has been widely explored as a therapeutic compound, and its application-alone or in combination with other molecules-is emerging as a potential effective strategy for the treatment and chemoprevention of tumours. In this study, we investigated the antitumor activity of AdoMet in Laryngeal Squamous Cell Carcinoma (LSCC), exploring the underlying mechanisms. (2) Results: We demonstrated that AdoMet induced ROS generation and triggered autophagy with a consistent increase in LC3B-II autophagy-marker in JHU-SCC-011 and HNO210 LSCC cells. AdoMet induced ER-stress and activated UPR signaling through the upregulation of the spliced form of XBP1 and CHOP. To gain new insights into the molecular mechanisms underlying the antitumor activity of AdoMet, we evaluated the regulation of miRNA expression profile and we found a downregulation of miR-888-5p. We transfected LSCC cells with miR-888-5p inhibitor and exposed the cells to AdoMet for 48 and 72 h. The combination of AdoMet with miR-888-5p inhibitor synergistically induced both apoptosis and inhibited cell migration paralleled by the up-regulation of MYCBP and CDH1 genes and of their targets. (3) Conclusion: Overall, these data highlighted that epigenetic reprogramming of miRNAs by AdoMet play an important role in inhibiting apoptosis and migration in LSCC cell lines.

Therapeutic Potential of the Natural Compound S-Adenosylmethionine as a Chemoprotective Synergistic Agent in Breast, and Head and Neck Cancer Treatment: Current Status of Research.

The present review summarizes the most recent studies focusing on the synergistic antitumor effect of the physiological methyl donor S-adenosylmethionine (AdoMet) in association with the main drugs used against breast cancer and head and neck squamous cell carcinoma (HNSCC), two highly aggressive and metastatic malignancies. In these two tumors the chemotherapy approach is recommended as the first choice despite the numerous side effects and recurrence of metastasis, so better tolerated treatments are needed to overcome this problem. In this regard, combination therapy with natural compounds, such as AdoMet, a molecule with pleiotropic effects on multiple cellular processes, is emerging as a suitable strategy to achieve synergistic anticancer efficacy. In this context, the analysis of studies conducted in the literature highlighted AdoMet as one of the most effective and promising chemosensitizing agents to be taken into consideration for inclusion in emerging antitumor therapeutic modalities such as nanotechnologies.

S-adenosylmethionine administration inhibits levodopa-induced vascular endothelial growth factor-A expression.

Background: Studies have demonstrated that S-adenosylmethionine could effectively affect the clinical wearing-off phenomena of levodopa, an antiparkinsonian agent; however, the detailed mechanisms for this effect need to be further clarified.Results: S-adenosylmethionine and levodopa had opposite effects on the protein stability of vascular endothelial growth factor-A. The analysis of tube formation and cell viability also showed the nonconforming functions of S-adenosylmethionine and levodopa on cell angiogenesis and proliferation. Meanwhile, S-adenosylmethionine could significantly abolish the increased angiogenesis and cell viability induced by levodopa. S-adenosylmethionine resulted in G1/S phase arrest, with decreased cyclin dependent kinase 4/6 and increased p16, a specific cyclin dependent kinase inhibitor. Mechanically, the different effects of levodopa and S-adenosylmethionine were dependent on the phosphorylation and activation of extracellular signal-regulated kinase. S-adenosylmethionine could be fitted into the predicted docking pocket in the crystal structure of vascular endothelial growth factor-A, enhancing its acetylation level and reducing half-life.Conclusions: These observations suggested that methyl donor S-adenosylmethionine could act as a potential agent against vascular endothelial growth factor-A-related diseases induced by levodopa treatment.Methods: We performed in vitro cytological analyses to assess whether S-adenosylmethionine intake could influence levodopa-induced vascular endothelial growth factor-A expression in human umbilical vein endothelial cells.

MiR-22, regulated by MeCP2, suppresses gastric cancer cell proliferation by inducing a deficiency in endogenous S-adenosylmethionine

This study investigated the effect of methyl-CpG-binding protein 2 (MeCP2) on miRNA transcription. Our results of miRNA chip assay and ChIP-seq showed that MeCP2 inhibited the expressions of numerous miRNAs by binding to their upstream elements, including not only the promoter but also the distal enhancer. Among the affected miRNAs, miR-22 was identified to remarkably suppress gastric cancer (GC) cell proliferation, arrest G1–S cell cycle transition, and induce cell apoptosis by targeting MeCP2, MTHFD2, and MTHFR. Understanding GC metabolism characteristics is the key to developing novel therapies that target GC metabolic pathways. Our study revealed that the metabolic profiles in GC tissues were altered. SAM (S-adenosylmethionine), a universal methyl donor for histone and DNA methylation, which is specifically involved in the epigenetic maintenance of cancer cells, was found increased. The production of SAM is promoted by the folate cycle. Knockdown of MTHFD2 and MTHFR, two key enzymes in folate metabolism and methyl donor SAM production, significantly suppressed GC cell proliferation. MiR-22 overexpression reduced the level of endogenous SAM by suppressing MTHFD2 and MTHFR, inducing P16, PTEN, and RASSF1A hypomethylation. In conclusion, our study suggests that miR-22 was inhibited by MeCP2, resulting in deficiency of endogenous SAM, and ultimately leading to tumor suppressor dysregulation.

Synthetase of the methyl donor S-adenosylmethionine from nitrogen-fixing α-rhizobia can bind functionally diverse RNA species

ABSTRACT Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated in vivo with MS2-tagged trans-sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia Sinorhizobium meliloti. The three proteomes were rather distinct, with that of EcpR1 particularly enriched in cell cycle-related enzymes, whilst sharing several transcription/translation-related proteins recurrently identified associated with sRNAs. Strikingly, MetK, the synthetase of the major methyl donor S-adenosylmethionine, was reliably recovered as a binding partner of the three sRNAs, which reciprocally co-immunoprecipitated with a FLAG-tagged MetK variant. Induced (over)expression of the trans-sRNAs and MetK depletion did not influence canonical riboregulatory traits, `for example, protein titration or sRNA stability, respectively. An in vitro filter assay confirmed binding of AbcR2, NfeR1 and EcpR1 to MetK and further revealed interaction of the protein with other non-coding and coding transcripts but not with the 5S rRNA. These findings uncover a broad specificity for RNA binding as an unprecedented feature of this housekeeping prokaryotic enzyme.

Serine Metabolism Controls Dental Pulp Stem Cell Aging by Regulating the DNA Methylation of p16

To investigate the characteristics and molecular events of dental pulp stem cells (DPSCs) for tissue regeneration with aging, we isolated and analyzed the stem cells from human exfoliated deciduous teeth (SHED) and permanent teeth of young (Y-DPSCs) and old (A-DPSCs) adults. Results showed that the stemness and osteogenic differentiation capacity of DPSCs decreased with aging. The RNA sequencing results showed that glycine, serine, and threonine metabolism was one of the most enriched gene clusters among SHED, Y-DPSCs, and A-DPSCs, according to analysis based on the Kyoto Encyclopedia of Genes and Genomes. The expression of serine metabolism–related enzymes phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate (PHGDH) decreased in A-DPSCs and provided less methyl donor S-adenosylmethionine (SAM) for DNA methylation, leading to the hypomethylation of the senescence marker p16 (CDNK2A). Furthermore, the proliferation and differentiation capacity of Y-DPSCs and SHED decreased after PHGDH siRNA treatment, which reduced the level of SAM. Convincingly, the ratios of PSAT1-, PHGDH-, or proliferating cell nuclear antigen–positive cells in the dental pulp of old permanent teeth were less than those in the dental pulp of deciduous teeth and young permanent teeth. In summary, the stemness and differentiation capacity of DPSCs decreased with aging. The decreased serine metabolism in A-DPSCs upregulated the expression of p16 via attenuating its DNA methylation, resulting in DPSC aging. Our finding indicated that serine metabolism and 1 carbon unit participated in stem cell aging, which provided new direction for stem cell aging study and intervention.

Cancer SLC43A2 alters T cell methionine metabolism and histone methylation.

Abnormal epigenetic patterns correlate with effector T cell malfunction in tumors1–4. However, their causal link is unknown. Here, we show that tumor cells disrupt methionine metabolism in CD8+ T cells, thereby lowering intracellular methionine levels and the methyl donor S-adenosylmethionine (SAM), resulting in loss of H3K79me2. Consequently, loss of H3K79me2 led to low STAT5 expression and impaired T cell immunity. Mechanistically, tumor cells avidly consumed and outcompeted T cells for methionine via high expression of SLC43A2, a methionine transporter. Genetic and biochemical inhibition of tumor SLC43A2 rescued T cell H3K79me2 levels, boosting spontaneous and checkpoint-induced tumor immunity. Moreover, we found that methionine supplementation improved expression of H3K79me2 and STAT5 in T cells, accompanied by increased T cell immunity in tumor bearing models and colon cancer patients. Clinically, tumor SLC43A2 negatively correlated with T cell histone methylation and functional gene signatures. Our work reveals a novel mechanistic connection between methionine metabolism, histone patterns, and T cell immunity in the tumor microenvironment. Thus, cancer methionine consumption is an unappreciated immune evasion mechanism, and targeting cancer methionine signaling may provide an immunotherapeutic approach.

Abstract 631: Mat2A Inhibitors decrease growth, increase senescence and p53 stability in MTAP-deleted cancer cells

Abstract Deletions in Methylthioadenosine phosphorylase (MTAP) are observed in 15% of all cancers including pancreatic, lung, bladder and GBM cancers (1). MTAP loss results in an accumulation of MTA which partially inhibits Protein Arginine Methyltransferase 5 (PRMT5). MTAP deletions confer enhanced sensitivity to inhibition of Methionine Adenosyltransferase 2A (Mat2A), which is involved in the synthesis of the universal methyl donor and PRMT5 substrate S-Adenosyl Methionine (SAM). In combination with MTA accumulation, Mat2A inhibition depletes SAM levels resulting in further inhibition of PRMT5 activity and a synthetic lethal (SL) relationship in MTAP null cancers. Inhibition of PRMT5 activity as a result increases RNA splicing defects and chromatin dysregulation which impacts tumor cell viability in different models. We developed MAT2A inhibitors for treatment of MTAP-deleted tumors, and in this study sought to determine the mechanism of action of these inhibitors in MTAP null models. These inhibitors were assessed for activity against both MTAP-wt and MTAP-null tumor models using cellular proliferation assays. MTAP-null (KP4, BxPC3, RT112/84, MiaPaCa-2, H647) models were more sensitive to Mat2A inhibition than MTAP-wt (HCT116, NCI-H460) models. Inhibition of Mat2A increased the expression of markers associated with cellular senescence and cell cycle arrest. Cellular SAM levels were diminished in both MTAP-wt and MTAP-null cells and xenografts while selective reduction of Symmetric Dimethyl Arginine (SDMA) levels were observed in MTAP-null models. Cellular response to Mat2A inhibitors is unaltered by RNAi-mediated knockdown of Mat1A. Signaling changes that were observed due to decreased PRMT5 activity included increased stability of p53, which may be due to the inefficient splicing of MDM4 mRNA, and was seen across multiple MTAP-null models but not in MTAP WT. Furthermore, increased protein and mRNA expression of the p53 target gene p21 was also observed in MTAP-null models. In conclusion, Mat2A inhibition effectively decreases SAM and SDMA levels, leading to increased stability of p53 and its targets and resulting in decreased cellular proliferation. Ongoing exploratory studies will further delineate the molecular mechanisms mediated by Mat2A inhibition in MTAP-null cell lines. The genes and metabolites identified from these studies will be validated in Mat2A-dependent and non-dependent MTAP-null cell lines identified from DepMap studies to inform further preclinical and clinical studies. Citation Format: Neil Bhola, Atieh Givmanesh, Marie-Claire Wagle, Candy Garcia, Kedar Vaidya, Leah Cleary, Zhonghua Pei, Mark Lackner, Zineb Mounir. Mat2A Inhibitors decrease growth, increase senescence and p53 stability in MTAP-deleted cancer cells [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 631.

Curcumin reduces methionine adenosyltransferase 2B expression by interrupting phosphorylation of p38 MAPK in hepatic stellate cells

The active polyphenol curcumin demonstrates therapeutic effects against various different diseases. Researches revealed the inhibitory roles of curcumin in hepatic stellate cell (HSC) activation and fibrogenesis. HSC activation, a key step in liver fibrogenesis, requires the remodeling of DNA methylation, which is associated with methionine adenosyltransferase II (MATII) composed of catalytic subunit MAT2A and regulatory subunit MAT2B. MATII is essential for HSC activation in vitro. The present researches aimed to investigate the effect of curcumin on MAT2B expression in HSCs in vivo and in vitro. Results demonstrated that curcumin could reduce MAT2B expression in HSCs at multiple levels. The activation of p38 MAPK pathway promoted MAT2B expression in HSCs. The effect of curcumin on MAT2B was through its interruption of p38 MAPK signaling pathway. Knockdown of MAT2B inhibited HSC activation and reduced collagen level in the model of liver fibrosis. Curcumin down-regulation of MAT2B contributed to the inhibitory role of curcumin on HSC activation and collagen expression in mouse livers. This study provided evidences for the effect of curcumin on the expression of MAT2B, an enzyme for the biosynthesis of methyl donor S-adenosylmethionine, in HSCs and demonstrated the function significance of curcumin-induced downregulation of MAT2B in curcumin inhibition of liver fibrosis.