Production Of S-adenosylmethionine Research Articles

Engineering of Halide Methyltransferase BxHMT through Dynamic Cross‐Correlation Network Analysis

AbstractHalide methyltransferases (HMTs) provide an effective way to regenerate S‐adenosyl methionine (SAM) from S‐adenosyl homocysteine and reactive electrophiles, such as methyl iodide (MeI) and methyl toluene sulfonate (MeOTs). As compared with MeI, the cost‐effective unnatural substrate MeOTs can be accessed directly from cheap and abundant alcohols, but shows only limited reactivity in SAM production. In this study, we developed a dynamic cross‐correlation network analysis (DCCNA) strategy for quickly identifying hot spots influencing the catalytic efficiency of the enzyme, and applied it to the evolution of HMT from Paraburkholderia xenovorans. Finally, the optimal mutant, M4 (V55T/C125S/L127T/L129P), exhibited remarkable improvement, with a specific activity of 4.08 U/mg towards MeOTs, representing an 82‐fold increase as compared to the wild‐type (WT) enzyme. Notably, M4 also demonstrated a positive impact on the catalytic ability with other methyl donors. The structural mechanism behind the enhanced enzyme activity was uncovered by molecular dynamics simulations. Our work not only contributes a promising biocatalyst for the regeneration of SAM, but also offers a strategy for efficient enzyme engineering.

Abstract PO5-08-06: Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells

Abstract Introduction. Oncogenic factors that are local/in-breast are of great interest as they may be more specifically targetable for breast cancer prevention than systemic factors. We have identified a lipid metabolism gene signature that is enriched in breast tissue at risk for estrogen negative breast cancer (ER- BC). Utilizing the medium chain fatty acid Octanoic acid (OA) to probe lipid metabolism in non-transformed breast epithelial cells, we observed increased flux through several metabolic reactions and altered histone methylation with consequent changes in gene expression (e.g. neural genes). Neuronal signaling and regulatory circuits are observed in cancer cells of multiple origins, not just ones with ontological relationships to neurons. We hypothesize that the first and rate limiting step in the de novo serine pathway, which is catalyzed by Phosphoglycerate Dehydrogenase (PHGDH) is key to these observations. In the forward direction, PHGDH participates in the serine, one-carbon, glycine (SOG) and methionine pathways that produce the methyl donor S-adenosylmethionine (SAM) and in the reverse direction produces the oncometabolite 2-Hydroxyglycerate (2-HG). Methods. Non-transformed MCF-10A cells exposed to OA were utilized for U13C-glucose tracing. SAM and 2-HG concentrations following treatment with OA ± PHGDH inhibitor were measured by liquid chromatography. CUT&RUN for H3K4me3 was performed and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. Single cell RNA-sequencing was carried out using breast microstructures derived from reduction mammoplasty tissue exposed to vehicle or OA. Microstructures were dissociated into single cells and sequenced using the 10x Genomics platform. The digital expression matrix file containing UMIs were analyzed with Seurat. Alkaline comet assay was performed to detect DNA breaks. Results. U13C-glucose tracing in presence of OA revealed that one-carbon-THF was redirected to the methionine cycle increasing flux to methylation. Concentrations of SAM and 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01), these genes are involved in neural pathways, EMT and ER- BC. Motif analysis revealed an overrepresentation of binding sites for transcription factors ATF3/4 (p < 0.05), which are regulators of the serine pathway. Single cell RNA-sequencing revealed OA not only affected the distribution of cell subpopulations but also modulated the expression of many genes within each subcluster. The percentage of luminal progenitor subcluster 3 increased upon OA from less than 1% to about 13%. Within basal subcluster 3, OA drives the expression of ATF3, along with two of the enzymes in the de novo serine pathway: PHGDH and PSAT1. Alkaline comet assay showed DNA breaks in OA- and control 2-HG- treated cells. Conclusions. Metabolism of OA in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM and 2-HG, with implications for oncogenesis: 1. SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express genes consistent with a neural/neural crest-like state. These co-opted neuronal regulatory mechanisms can make critical contributions to the acquired functional capabilities that drive cancer development. 2. 2-HG exposure results in appearance of DNA breaks, which are likely consequent to the inhibition of the alpha-ketoglutarate-dependent dioxygenases KDM 4A/B by 2-HG. Their catalytic activity is required for homologous recombination repair; inhibition results in metabolic “BRCAness”. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Maria Paula Zappia, Elizaveta V. Benevolenskaya, Abul Bashar Mir Md. Khademul Islam, Maxim V. Frolov, Seema Khan, Susan Clare. Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-08-06.

Development and validation of a generic methyltransferase enzymatic assay based on an SAH riboswitch

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) – the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAH. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z’ > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

Engineering of Halide Methyltransferase BxHMT through Dynamic Cross-Correlation Network Analysis.

Halide methyltransferases (HMTs) provide an effective way to regenerate S-adenosyl methionine (SAM) from S-adenosyl homocysteine and reactive electrophiles, such as methyl iodide (MeI) and methyl toluene sulfonate (MeOTs). As compared with MeI, the cost-effective unnatural substrate MeOTs can be accessed directly from cheap and abundant alcohols, but shows only limited reactivity in SAM production. In this study, we developed a dynamic cross-correlation network analysis (DCCNA) strategy for quickly identifying hot spots influencing the catalytic efficiency of the enzyme, and applied it to the evolution of HMT from Paraburkholderia xenovorans. Finally, the optimal mutant, M4 (V55T/C125S/L127T/L129P), exhibited remarkable improvement, with a specific activity of 4.08 U/mg towards MeOTs, representing an 82-fold increase as compared to the wild-type (WT) enzyme. Notably, M4 also demonstrated a positive impact on the catalytic ability with other methyl donors. The structural mechanism behind the enhanced enzyme activity was uncovered by molecular dynamics simulations. Our work not only contributes a promising biocatalyst for the regeneration of SAM, but also offers a strategy for efficient enzyme engineering.

Abstract 444: Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells

Abstract Introduction. A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increases flux through various metabolic reactions including those involved in serine, one-carbon, glycine (SOG) and methionine. We hypothesized that the metabolism of lipids in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing S-adenosylmethionine (SAM) leading to histone methylation increases and changes in gene expression. Methods. MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± the PHGDH inhibitor CBR-5884. ROS-induced redox changes were monitored live in cells transduced with ORP1-roGFP2 vectors. CUT&amp;RUN was performed for H3K4me3. Expression of OA-induced genes was measured by rt-qPCR upon exposure to OA ± CBR-5884 or OA ± the histone methyltransferase (HMT) inhibitor Piribedil. Alkaline comet assay was done to detect DNA breaks. Single-cell RNA-seq (scRNAseq) was performed on tissue-derived breast microstructures exposed to ± OA. Results. Upon OA treatment, the Cancer index increased and 1C-THF was redirected to the methionine cycle increasing flux to methylation. OA significantly increased the production of SAM, GSH and 2-HG after 15 min. Blocking the first enzyme in the serine pathway, PHGDH, prevented these increases. After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p &amp;lt; 0.0001), peaking at 15 min. H3K4me3 CUT&amp;RUN revealed 661 differential peaks (FDR &amp;lt; 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR &amp;lt; 0.01) including neural, EMT and ER- BC related genes. Motif analysis revealed an overrepresentation of binding sites for serine pathway transcriptional activators ATF3/4 (p &amp;lt; 0.05). Blocking PHGDH or HMT prevented OA-induced gene expression changes. Alkaline comet assay showed that OA significantly increased comet tails. scRNAseq revealed OA increased the numbers of cells within all cell clusters expressing ATF3 and PHGDH. Also observed was an increase in the percentage of basal BSL1, luminal progenitor LP3 and hormone sensing HS1 cells. Conclusions. Metabolism of OA results in a metabolic shift toward the SOG and methionine increasing the production of SAM, GSH and 2-HG which have implications for oncogenesis. The increased SAM fosters epigenetic phenotypic plasticity via altered histone methylation. The increased proportion of specific cell types likely reflects the survival of cells able to mitigate oxidative stress. Increased 2-HG may produce metabolic BRCAness by inhibiting 2-oxoglutarate-dependent dioxygenases. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta V. Benevolenskaya, Maxim V. Frolov, Jason Locasale, Marcelo G. Bonini, Navdeep S. Chandel, Seema Khan, Susan Clare. Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 444.

MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis-infected human gingival fibroblasts

ABSTRACT Background Methionine adenosyl transferase II alpha (MAT2A) is the key enzyme to transform methionine into S-adenosylmethionine (SAM), the main methylgroup donor involved in the methylation. The purpose of our study wasto explore whether MAT2A-mediated methionine metabolism affected theexpression of inflammatory cytokines in human gingival fibroblasts(hGFs). Methods Both healthy and inflamed human gingiva were collected. HGFs werecultured and treated with P. gingivalis, with or without MAT2Ainhibitor (PF9366), small interference RNA (siRNA), or extrinsic SAMpretreatment. The levels of inflammatory cytokines were detected byreal-time PCR, western blotting, and ELISA. SAM levels were detectedby ELISA. The nuclear factor-kappa B (NF-κB) and mitogen-activatedprotein kinase (MAPK) pathway was explored by western blotting. Results The expression of MAT2A was increased in the inflamed tissues. P.gingivalis infection promoted the expression of MAT2A and SAM inhGFs. Meanwhile, PF9366 and MAT2A-knockdown significantly decreasedexpression of inflammatory cytokines and SAM production. PF9366inhibited activation of NF-κB/MAPK pathway in P. gingivalis-treatedhGFs. Conclusions MAT2A-mediated methionine metabolism promoted P. gingivalis-inducedinflammation in hGFs. Targeting MAT2A may provide a novel therapeuticmethod for modulating periodontitis.

Single Cell Multi-Omic Analysis of Neoplastic Plasma Cells across the Disease Spectrum Identifies Novel Pathobiologic Mediators and Potential Therapeutic Targets in Multiple Myeloma (MM)

Background: A challenge to developing a full understanding of MM pathobiology using bulk profiling techniques is the large degree of intra-tumoral heterogeneity from minor subclones that, despite their low representation, nonetheless contribute to therapy resistance and disease progression. Moreover, our current therapies tend to be targeted against pathways common to both neoplastic and normal plasma cells (PCs), indicating a need to define novel tumor biology modifiers that could potentially serve to improve risk stratification and as therapeutic targets. Methods: To overcome these limitations, we simultaneously evaluated individual PC single-cell transcriptome and B-cell receptor variable (VDJ) sequences from freshly enriched bone marrow biopsies. These included 33 patients with precursor conditions, 17 each with newly diagnosed symptomatic or relapsed/refractory disease (RRMM), and 3 controls using the 10x Genomics platform. A total of ~320,000 PCs were analyzed, yielding median per sample cell counts of 4,626 and ~300 million total reads. Identical immunoglobulin VDJ sequences defined monoclonal PCs, while diverse VDJ sequences at low frequencies defined polyclonal cells, allowing each patient to serve as their own control. Results: The percentage of monoclonal PCs in active MM was &amp;gt;84%, while precursor stages were more variable (40-100% in smoldering MM; 0.3-82% in monoclonal gammopathy of undetermined significance (MGUS)). Though we could not identify monoclonal PCs in two MGUS cases, we identified them by scRNA-seq in four MGUS patients whose bone marrow showed no clonal cells by standard clinical multiparametric flow cytometry. In differential gene expression analysis where samples were grouped based on the diagnosis, 501 dysregulated genes were identified in monoclonal PCs from symptomatic MM compared to polyclonal PCs. These included genes previously identified as disease modifiers, including Hepatocyte growth factor, Lactate dehydrogenase A, Dickkopf WNT signaling pathway inhibitor 1, CD27, and Lysosomal associated membrane protein family member 5, providing validation for our approach. Next, we identified 279 genes that were commonly dysregulated in monoclonal PCs from symptomatic vs. asymptomatic MM. We also performed unsupervised hierarchical clustering of the top 1000 most variable genes and defined three diagnostic categories. Significant ontology terms characterizing these categories broadly involved antigen processing and recognition in Group 1 (enriched in precursor stages); transcription and translation control in Group 2 (enriched in active MM); and cell division and DNA metabolism in Group 3 (enriched with RRMM) ( A). We focused on dysregulated targets that: showed differential expression in monoclonal vs. polyclonal PCs; expression in most patients within a diagnostic category; a correlation with outcomes in the CoMMpassstudy; and novelty in MM. Among these were Mitotic arrest deficient 2 like 1 (MAD2L1), a component of the mitotic spindle assembly checkpoint, and Methionine adenosyltransferase 2A (MAT2A), which catalyzes the production of S-adenosylmethionine, a key methyl donor in multiple cellular processes, from methionine and ATP. Notably, MAD2L1 inhibition with M2I1, and MAT2A targeting with the allosteric inhibitor AG-270, both produced a dose- and time-dependent inhibition in cell proliferation and reduction in myeloma cell line viability. Importantly, the effects of AG-270 ( B) were associated with induction of markers of apoptosis, including Caspase cleavage, and a reduction of Histone H3 dimethylation. Moreover, they occurred at clinically relevant concentrations based on the known pharmacokinetics of this drug in Phase I testing. Conclusions: Our combined single-cell transcriptomics and VDJ sequencing allowed a greater understanding of heterogeneity among neoplastic PCs compared to each patient's own polyclonal PCs. These analyses can identify putative novel mediators of disease pathobiology that impact prognosis, and that can serve as potential new therapeutic targets using either novel agents, or drug repurposed from other therapeutic areas, setting the stage for their translation to the clinic.

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism.

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

Engineering of Methionine Adenosyltransferase Reveals Key Roles of Electrostatic Interactions in Enhanced Catalytic Activity.

As an important dietary supplement, S-adenosylmethionine (SAM) is currently synthesized by methionine adenosyltransferase (MAT) using ATP and methionine as substrates. However, the activity of MAT is severely inhibited by product inhibition, which limits the industrial production of SAM. Here, MAT from Bacteroides fragilis (BfMAT), exhibiting relatively low product inhibition and moderate specific activity, was identified by gene mining. Based on molecular docking, residues within 5Å of ATP in BfMAT were subjected to mutagenesis for enhanced catalytic activity. Triple variants M3-1 (E42M/E55L/K290I), M3-2 (E42R/E55L/K290I), and M3-3 (E42C/E55L/K290I) with specific activities of 1.83, 1.81, and 1.94 U/mg were obtained, which were 110.5-125.6% higher than that of the wild type (WT). Furthermore, compared with WT, the Km values of M3-1 and M3-3 were decreased by 31.4% and 60.6%, leading to significant improvement in catalytic efficiency (kcat/Km) by 322.5% and 681.1%. All triple variants showed shifted optimal pH from 8.0 to 7.5. Moreover, interaction analysis suggests that the enhanced catalytic efficiency may be attributed to the decreased electrostatic interactions between ATP and the mutation sites (E42, E55, and K290). Based on MD simulation, coulomb energy and binding free energy analysis further reveal the importance of electrostatic interactions for catalytic activity of BfMAT, which could be an efficient strategy for improving catalytic performance of MATs.

Abstract 1637: MAT2A inhibition in MTAP-/- tumors confers mechanistic vulnerabilities to multiple clinically actionable synthetic lethal drug combinations

Abstract Methylthioadenosine phosphorylase (MTAP) is co-deleted with the tumor suppressor CDKN2A in approximately 15% of solid tumors and can elicit dependency on methionine adenosyltransferase 2A (MAT2A) production of S-adenosyl methionine (SAM), the major methyl donor for cellular methyltransferase reactions. IDE397, a potent small molecule inhibitor of MAT2A, was developed to exploit this synthetic lethal relationship for therapeutic benefit in patients harboring MTAP-/- cancers and demonstrates selective anti-tumor activity in MTAP-/- models. Evaluation of IDE397 efficacy across a panel of 48 MTAP-/- PDX models revealed consistent tumor growth inhibition (TGI&amp;gt;60%) across diverse lineages, with distinct enrichment of tumor regressions in squamous lung cancers. To further enhance the efficacy of IDE397 within and among tumor lineages, we sought to identify rational combination partners. A three-pronged approach was employed that dovetailed molecular profiling of IDE397 drug effects in vivo, chemogenomic evaluation of selective drug sensitivities in MTAP-/- cell lines across the CCLE, and high-throughput in vitro drug combination screens. Unbiased pathway analysis of IDE397-dependent changes in MTAP-/- PDX transcriptomes revealed perturbations in pre-mRNA splicing, DNA damage repair, and mitotic spindle assembly as commonly enriched across diverse models. Evaluation of altered pre-mRNA splicing patterns suggested that many of these changes may be a consequence of selective impairment of PRMT5-dependent support of spliceosome activity. Notably, sensitivities to drugs that inhibit the fidelity of pre-mRNA splicing, genome stability, and microtubule stability were enriched in MTAP-/- cell lines, suggesting that the biological processes selectively altered by IDE397 in tumors are already partially impaired by MTAP deletion. Finally, an IDE397 in vitro drug combination viability screen of over 400 drugs returned taxanes, platins, topoisomerase inhibitors, splicing inhibitors, and anti-folates as selectively synergistic with IDE397 in MTAP-/- models. Synergy between IDE397 and pemetrexed was of particular interest given mechanistic convergence of the methionine salvage and folate cycle pathways on nucleotide synthesis in the context of MTAP-/-. Accordingly, this combination provided enhanced tumor growth inhibition in vivo in MTAP-/- NSCLC and bladder models and is currently under evaluation in phase 1. Collectively, these observations indicate that MAT2A inhibition can generate cell states in MTAP-/- tumor cells that are selectively vulnerable to approved chemotherapies and targeted therapies. These synergistic relationships may provide a predictive biomarker strategy for multiple IDE397 synthetic lethal combination therapies. Citation Format: Kimberline Y. Gerrick, Jenny Laraio, Kelsey Annen, Hoang Tran, Marcus M. Fischer, Yevgeniy Freyman, Geeta Sharma, Isaac Bishof, Stephen Federowicz, Divya Pankajakshan, Claire L. Neilan, Biju Mangatt, Anthony Mazurek, Mark R. Lackner, Michael White, Zineb Mounir. MAT2A inhibition in MTAP-/- tumors confers mechanistic vulnerabilities to multiple clinically actionable synthetic lethal drug combinations [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 1637.

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.

Molecular Identification, Dimorphism and Virulence of C. albicans

C. albicans causes human diseases, especially in immune-compromised patients. The current study aimed to identify Candida albicans using different techniques. Dimorphism and virulence behaviour were also studied. A Candida albicans strain was firstly identified by biochemical methods using VITEK 2 Compact automated technique and chromogenically using CHROMagar differential media that differentiate between Candida spp. Based on an enzymatic reaction. Molecular identification using ITS primers was also used to confirm Candida albicans identification. Accession number of the identified C. albicans was obtained as OK104215. The enhancement of dimorphism was studied using RPMI 1640 media (Roswell Park Memorial Institute Medium), while monitoring growth at different time intervals under microscope to investigate dimorphic changes. C. albicans showed its optimum dimorphism after 36-66 hours at 37◦C. HPLC analysis for the enzyme product S-adenosylmethionine (SAM) was carried out at different time intervals. By increasing time, SAM production increased until optimum production reached after 72h of incubation on RPMI 1640. After that the production of SAM began to decrease.

Enhanced selenocysteine biosynthesis for seleno-methylselenocysteine production in Bacillus subtilis.

Seleno-methylselenocysteine (SeMCys) is an effective component for selenium supplementation with anti-carcinogenic potential and can ameliorate neuropathology and cognitive deficits. In this study, we aimed to engineer Bacillus subtilis 168 for the microbial production of SeMCys. First, the accumulation of intracellular selenocysteine (SeCys) as the precursor of SeMCys was enhanced through overexpression of serine O-acetyltransferase, which was desensitized against feedback inhibition by cysteine. Next, the S-adenosylmethionine (SAM) synthetic pathway was optimized to improve methyl donor availability through expression of S-adenosylmethionine synthetase. Further, SeMCys was successfully produced through expression of the selenocysteine methyltransferase in SeCys and SAM-producing strain. The increased expression level of selenocysteine methyltransferase benefited the SeMCys production. Finally, all the heterologous genes were integrated into the genome of B. subtilis, and the strain produced SeMCys at a titer of 18.4μg/L in fed-batch culture. This is the first report on the metabolic engineering of B. subtilis for microbial production of SeMCys and provides a good starting point for future pathway engineering to achieve the industrial-grade production of SeMCys. KEY POINTS: • Expression of the feedback-insensitive serine O-acetyltransferase provided B. subtilis the ability of accumulating SeCys. • SAM production was enhanced through expressing S-adenosylmethionine synthetase in B. subtilis. • Expression of selenocysteine methyltransferase in SeCys and SAM-accumulating strain facilitated SeMCys production.

Abstract P6-11-03: Lipid-rich environment induces epigenetic reprogramming in non-transformed breast epithelial cells enhancing a mammary cell plasticity

Abstract Introduction. The identification of women specifically at risk for estrogen receptor negative breast cancer (ER- BC) and the targeted treatment of this disease are significantly unmet clinical needs. We analyzed the gene expression profiles of epithelial cells from the contralateral unaffected breasts (CUBs) of BC patients and identified a lipid metabolism gene signature enriched in the CUBs of women with ER- BC (PMID: 28263391). Subsequently, we observed that exposure of non-transformed breast epithelial cells to lipids results in significant changes in gene expression and histone posttranslational modifications; and increased flux through multiple metabolic reactions, including those of serine and methionine (PMID: 35508495). Interestingly, the upregulated genes are involved in neural development, axon guidance, neural crest pathways and stemness. Neural genes are highly expressed in Triple Negative Breast Cancers (TNBCs) especially in the C2 cluster. Given that mammary stem/progenitor cells have distinct metabolic properties compared to other mammary cell subsets, we hypothesized that upon lipid exposure, stem-like cells have a survival advantage, and that lipid induces epigenetic reprogramming into neural-like cells which may foster a malignant transformation. Methods. To interrogate potential mechanisms linking lipids and epigenetic reprogramming, we performed CUT&amp;RUN for H3K4me3 and H3K27me3 in non-transformed, estrogen and progesterone receptor negative MCF-10A cells cells exposed to vehicle or octanoic acid (OA) for 24 hrs. Peaks were called using MACS2 and differential peaks identified with DiffBind. Differentially expressed genes affected by OA (PMID: 28263391) were compared with target genes from the CUT&amp;RUN. To determine the contribution in OA exposed cells of serine and methionine metabolism to S-adenosyl methionine (SAM), 13C-glucose tracing was performed. The Aldefluour assay was used to identify stem-like (ALDH+) cells in lipid-exposed MCF-10A cells. To determine if lipid-exposed cells adopt a neural-like phenotype, MCF-10A cells were grown on Poly-D-Lysine/Laminin (PDL/LM) coated plates. Results. A total of 661 differential peaks were identified for H3K4me3 (FDR &amp;lt; 0.05) encompassing TNBC C2 markers (NRTN, CHRM3) and genes involved in neuron differentiation, axonogenesis (NGFR, NGF, FOXA2), neural crest migration (NTRK2, MMP2) and EMT (DLL4, MMP15). Approximately 73% of H3K4me3 OA-associated peaks encompass regulatory regions of genes that experienced a significant increase in gene expression with OA exposure (FDR &amp;lt; 0.01), including NGFR (log2 FC = 11.7), FOXA2 (log2 FC = 11.6), NGF (log2 FC = 8). Twelve H3K27me3 peaks were significantly enriched in vehicle (FDR &amp;lt; 0.05) and associated with increased gene expression in OA, among them were the stem cell markers LGR6 (log2 FC = 1.9) and PLAG1 (log2 FC = 2.8). 13C stable isotope tracing revealed that in presence of OA, glucose contributes to increased fractional abundance of the SAM M+1 isotopologue (adj p = 0.003) indicating that carbons derived from the serine synthesis pathway are used for re-methylation of homocysteine to methionine. Vehicle treated cells growing on PDL/LM plates assumed an epithelial phenotype while OA-treated cells adopted a neurite-like phenotype. Upon OA treatment the percentage of ALDH+ cells increased by a minimum of 10%. Conclusions. The increase of fractional abundance of SAM and the upregulation of neural genes regulated by H3K4me3 as well as the enrichment of ALDH+ cells and the development of a neural-like phenotype in a rich lipid environment, suggests that lipid exposure affects the production of SAM, 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. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Seema Khan, Susan Clare. Lipid-rich environment induces epigenetic reprogramming in non-transformed breast epithelial cells enhancing a mammary cell plasticity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-03.

Utilization of high-K+-cane molasses for enhanced S-Adenosylmethionine production by manipulation of a K+ transport channel in Saccharomyces cerevisiae

S-Adenosylmethionine (SAM) is a valuable compound extensively used in depression treatment and biological methyl donor, which is biosynthesized from ATP and L-methionine with SAM synthase (SAM-s) as a catalyst. In this research, the weak SAM-s activity was a key factor and led to low SAM titer in Saccharomyces cerevisiae. Then, a novel K+ uptake channel was introduced to the SAM production pathway of Saccharomyces cerevisiae, activated by K+ in molasses to enhance the SAM synthase (SAM-s) activity and thereby improve the production efficiency of SAM. As a result, the intracellular K+ concentration and SAM-s activity were 1.23- and 1.31-fold higher than the control leading to a 24 % increase to 0.93 ± 0.04 g/L in SAM titer. In combination with bioprocess optimization resulted in a final yield of 2.22 ± 0.01 g/L in shake flasks and 15.37 ± 0.89 g/L in a 5-L bioreactor from pretreated molasses. This work provides a practical and cost-effective strategy for the large-scale biosynthesis of SAM from cane molasses.

Development of a synthetic transcription factor-based S-adenosylmethionine biosensor in Saccharomyces cerevisiae.

S-Adenosylmethionine (SAM) is a crucial small-molecule metabolite widely used in food and medicine. The development of high-throughput biosensors for SAM biosynthesis can significantly improve the titer of SAM. This paper constructed a synthetic transcription factor (TF)-based biosensor for SAM detecting in Saccharomyces cerevisiae. The synthetic TF, named MetJ-hER-VP16, consists of an Escherichia coli-derived DNA-binding domain MetJ, GS linker, the human estrogen receptor binding domain hER, and the viral activation domain VP16. The synthetic biosensor is capable of sensing SAM in a dose-dependent manner with fluorescence as the output. Additionally, it is tightly regulated by the inducer SAM and β-estradiol, which means that the fluorescence output is only available when both are present together. The synthetic SAM biosensor could potentially be applied for high-throughput metabolic engineering and is expected to improve SAM production.

Untargeted Metabolomics Combined with Metabolic Flux Analysis Reveals the Mechanism of Sodium Citrate for High S-Adenosyl-Methionine Production by Pichia pastoris

S-adenosyl-methionine (SAM) is crucial for organisms to maintain some physiological functions. However, the inconsistency between high L-methionine feeding rate and yield during SAM production at an industrial scale and its metabolic mechanism have not been elucidated. Here, the cellular metabolic mechanism of feeding sodium citrate to the Pichia pastoris (P. pastoris) G12’/AOX-acs2 strain to enhance SAM production was investigated using untargeted metabolomics and metabolic flux analysis. The results indicated that the addition of sodium citrate has a facilitative effect on SAM production. In addition, 25 metabolites, such as citrate, cis-aconitate, and L-glutamine, were significantly up-regulated, and 16 metabolites, such as glutathione, were significantly down-regulated. Furthermore, these significantly differential metabolites were mainly distributed in 13 metabolic pathways, such as the tricarboxylic acid (TCA) cycle. In addition, the metabolic fluxes of the glycolysis pathway, pentose phosphate pathway, TCA cycle, and glyoxylate pathway were increased by 20.45–29.32%, respectively, under the condition of feeding sodium citrate compared with the control. Finally, it was speculated that the upregulation of dihydroxyacetone level might increase the activity of alcohol oxidase AOX1 to promote methanol metabolism by combining metabolomics and fluxomics. Meanwhile, acetyl coenzyme A might enhance the activity of citrate synthase through allosteric activation to promote the flux of the TCA cycle and increase the level of intracellular oxidative phosphorylation, thus contributing to SAM production. These new insights into the L-methionine utilization for SAM biosynthesis by systematic biology in P. pastoris provides a novel vision for increasing its industrial production.

Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents

BackgroundOne-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids.ResultsWe describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, β-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels.ConclusionsThis work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

O-302 Lower expression of AHCY is associated with increased miscarriage risk

Abstract Study question Are alterations in the transcript levels of genes from the methionine cycle associated with maternal associated miscarriage risk? Summary answer Lower expression of AHCY is associated with a greater number of prior pregnancy losses What is known already Around 15% of pregnancies end in miscarriage, and the risk of recurrence increases with each pregnancy loss. Aberrant differentiation (decidualization) of endometrial stromal cells into specialised decidual cells to accommodate implantation is a key maternal factor for miscarriage risk. Our previous work identified secretory changes in cysteine and methionine metabolites upon decidualization. The methionine cycle contributes to vital cellular functions, including producing methionine for proliferation, regulating cell differentiation, and S-adenosylmethionine (SAM) production. SAM is required for protein, RNA and DNA methylation, thereby influencing pathways at the metabolic, epigenetic, and proteomic levels. AHCY clears S-adenosylhomocysteine (SAH), reducing its inhibition of methylation. Study design, size, duration Endometrial biopsies (n = 250) were collected during the luteal phase (LH + 6-11). Patients were grouped based on their miscarriage history. Accordingly, expression of genes from the methionine cycle were quantified using RT-qPCR. Participants/materials, setting, methods Endometrial biopsies were obtained, with written informed consent, from women attending the Implantation Clinic at University Hospitals Coventry and Warwickshire NHS Trust, following transvaginal ultrasounds to exclude uterine pathology. Isolated RNA was converted into cDNA. Expression of AHCY, AMD1, BHMT2, CBS, MAT2A, MAT2B, and MTR were normalised to L19. Statistical analysis was performed in Graphpad Prism; with significance accepted at p &amp;lt; 0.05. AHCY was silenced in an endometrial cell line to determine its effect on decidualization. Main results and the role of chance This study reports a distinct reduction in expression of methionine cycle genes (MAT2A, AHCY, AMD1, MTR, BHMT2) in the late-luteal phase of the cycle consistent with a reduction in proliferation. By plotting percentile graphs based on the statistical distribution in gene expression for each day of the luteal phase, comparisons have been made between groups. AHCY expression is significantly reduced in patients with increasing number of prior miscarriages, particularly between 0-2 and 5+ previous miscarriages (p = 0.0334). Neither patient age nor BMI are a factor in this reduced expression. In contrast, there is a stromal specific increase in AHCY upon decidualization in vitro, suggesting it is required in the decidua. Silencing AHCY in an endometrial cell line significantly reduces PRL expression upon decidualization. Reduction in AHCY may lead to a decreased “methylation potential” as SAH cannot be cleared. SAH accumulation inhibits methylation, and limits SAM production, thus compounding its effect. Decreased methylation potential could prevent differentiation of the stromal compartment, resulting in lower levels of PRL, and altering decidual timing. Therefore, an embryo may implant into a tissue primed for disintegration, resulting a miscarriage. In summary, AHCY may contribute to aberrant decidualization augmenting the risk of miscarriage. Limitations, reasons for caution Results are based on endometrial biopsies from an implantation clinic, therefore studies into biopsies from women with normal reproductive histories should also be analysed. Further functional studies are needed to ascertain the mechanism of action of AHCY in miscarriage. Wider implications of the findings This study identified that a decrease in AHCY in whole endometrial tissue is associated with increased risk of miscarriage. Further, silencing AHCY perturbed decidual marker expression. Thus, AHCY may act as a biomarker for atypical decidualization, and the clearance of SAH may be a potential treatment. Trial registration number N/A