Nicotinamide N-methyltransferase Research Articles

Nicotinamide N-Methyltransferase Expression in Cervical Adenocarcinoma and Its Clinical Significance

Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme increasingly implicated in tumor progression and resistance to treatment. It is overexpressed in human cancers including glioblastoma, gastric, pancreatic, lung and colorectal cancers. The role of NNMT in cervical adenocarcinoma has not been studied thus far. We aimed to evaluate expression of NNMT in cervical adenocarcinoma and investigate its prognostic significance. NNMT expression was assayed in 18 cases of cervical adenocarcinoma and 19 cases of benign cervical tissue. NNMT immunoreactivity was scored by multiplying staining intensity with percentage of immunoreactive cells. The expression of NNMT was significantly higher in cervical adenocarcinoma than in benign tissue (p = 0.024). NNMT expression was significantly higher in patients with locally advanced than early cervical cancer (p = 0.031). NNMT expression was higher in patients with tumor size greater than 4 cm and in those with poorly differentiated tumors compared to patients with tumors of 4 cm or less and well-moderate differentiation, respectively (p = 0.044, p = 0.014). Although on a preliminary level, this is the first study to detect an overexpression of NNMT in cervical adenocarcinoma and an increased expression associated with advanced stage, increased tumor size and poor differentiation. NNMT has been shown to carry prognostic and therapeutic importance in a variety of cancers, and it can be a potential treatment target in cervical adenocarcinomas.

Identification of evolutionary and kinetic drivers of NAD-dependent signaling

Nicotinamide adenine dinucleotide (NAD) provides an important link between metabolism and signal transduction and has emerged as central hub between bioenergetics and all major cellular events. NAD-dependent signaling (e.g., by sirtuins and poly-adenosine diphosphate [ADP] ribose polymerases [PARPs]) consumes considerable amounts of NAD. To maintain physiological functions, NAD consumption and biosynthesis need to be carefully balanced. Using extensive phylogenetic analyses, mathematical modeling of NAD metabolism, and experimental verification, we show that the diversification of NAD-dependent signaling in vertebrates depended on 3 critical evolutionary events: 1) the transition of NAD biosynthesis to exclusive usage of nicotinamide phosphoribosyltransferase (NamPT); 2) the occurrence of nicotinamide N-methyltransferase (NNMT), which diverts nicotinamide (Nam) from recycling into NAD, preventing Nam accumulation and inhibition of NAD-dependent signaling reactions; and 3) structural adaptation of NamPT, providing an unusually high affinity toward Nam, necessary to maintain NAD levels. Our results reveal an unexpected coevolution and kinetic interplay between NNMT and NamPT that enables extensive NAD signaling. This has implications for therapeutic strategies of NAD supplementation and the use of NNMT or NamPT inhibitors in disease treatment.

Elevated N‐methyltransferase expression induced by hepatic stellate cells contributes to the metastasis of hepatocellular carcinoma via regulation of the CD44v3 isoform

The cross‐talk between hepatic stellate cells (HSCs) and hepatic carcinoma cells contributes to hepatocellular carcinoma (HCC) progression, but the underlying mechanism is largely unknown. We report here that activated HSCs induce upregulation of nicotinamide N‐methyltransferase (NNMT), which is known to regulate multiple metabolic pathways in hepatoma cells of the liver. High levels of NNMT in HCC tissues were positively correlated with vascular invasion, increased serum HBV‐DNA levels, and distant metastasis. In addition, functional assays showed that NNMT promoted HCC cell invasion and metastasis by altering the histone H3 methylation on 27 methylation pattern and transcriptionally activating cluster of differentiation 44 (CD44). NNMT‐mediated N6‐methyladenosine modification of CD44 mRNA resulted in the formation of a CD44v3 splice variant, while its product 1‐methyl‐nicotinamide stabilized CD44 protein by preventing ubiquitin‐mediated degradation. Finally, NNMT was also shown to be a target of statins that inhibited metastasis of hepatoma cells. Taken together, our study shows for the first time that the NNMT/CD44v3 axis regulates HCC metastasis and presents NNMT as a promising prognostic biomarker and therapeutic target for HCC.

Downregulation of nicotinamide N-methyltransferase inhibits migration and epithelial-mesenchymal transition of esophageal squamous cell carcinoma via Wnt/β-catenin pathway.

Nicotinamide N-methyltransferase (NNMT) is an important methyltransferase involved in the biotransformation of many drugs and exogenous compounds. Abnormal expression of NNMT protein is closely associated with the onset and progression of many malignancies, but little is known about its role in esophageal squamous cell carcinoma (ESCC). Therefore, we aimed to explore whether NNMT plays any roles in carcinogenesis and metastasis in ESCC. NNMT expression was determined by immunohistochemistry in ESCC and corresponding adjacent normal tissues. Functional experiments were performed to elucidate the effects of NNMT knockdown on the proliferation, apoptosis, cell cycle, migration, and epithelial-mesenchymal transition (EMT) in EC9706 and TE1 cells. NNMT expression was significantly elevated in ESCC tissues compared with corresponding adjacent normal tissues. Moreover, a significant association emerged between NNMT expression and lymph node metastasis. SiRNA-mediated knockdown of NNMT in ESCC cells can significantly suppress cell viability and migration, induce cell cycle arrest, and promote cell apoptosis. In addition, NNMT downregulation led to the reversal of EMT, as reflected by upregulation of the intercellular adhesion molecule E-cadherin and downregulation of the mesenchymal markers N-cadherin and Vimentin. Further study found that NNMT knockdown suppressed the Wnt/β-catenin signaling pathway. Taken together, these findings indicate that NNMT is a critical regulator of EMT in ESCC and may be a potential therapeutic target for ESCC metastasis.

Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT) with Enhanced Activity.

Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide to form N-methylnicotinamide. Overexpression of NNMT is associated with a variety of diseases, including a number of cancers and metabolic disorders, suggesting a role for NNMT as a potential therapeutic target. By structural modification of a lead NNMT inhibitor previously developed in our group, we prepared a diverse library of inhibitors to probe the different regions of the enzyme’s active site. This investigation revealed that incorporation of a naphthalene moiety, intended to bind the hydrophobic nicotinamide binding pocket via π–π stacking interactions, significantly increases the activity of bisubstrate-like NNMT inhibitors (half-maximal inhibitory concentration 1.41 μM). These findings are further supported by isothermal titration calorimetry binding assays as well as modeling studies. The most active NNMT inhibitor identified in the present study demonstrated a dose-dependent inhibitory effect on the cell proliferation of the HSC-2 human oral cancer cell line.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

NNMT Involvement in CD44v3 Regulation Induced by Hepatic Stellate Cells Contributes to the Metastasis of Hepatocarcinoma Cells

The crosstalk between hepatic stellate cells (HSCs) and hepatic carcinoma cells contributes to hepatocellular carcinoma (HCC) progression, although the underlying mechanism is largely unknown. We found that activated HSCs upregulated nicotinamide (NAM) N-methyltransferase (NNMT) expression in hepatoma cells. NNMT regulates multiple metabolic pathways in the liver, and aberrantly high levels of NNMT in the HCC tissues were positively correlated with vascular invasion, serum HBV levels and distant metastasis in our study. Functional assays showed that NNMT promoted cell migration, invasion and metastasis in vitro and in vivo. Mechanistically, NNMT regulated the intracellular methylation potential which in turn altered histone H3K27 methylation pattern, resulting in the transcriptional activation of CD44. NNMT-mediated m6A modification of the CD44 mRNA resulted in the formation of the CD44v3 splice variant, while its product 1-MNA stabilized CD44 protein by preventing ubiquitin-mediated degradation. Finally, NNMT was also a target of statins which inhibited the metastatic ability of hepatoma cells. Taken together, our study provides evidence for the first time on the potential role of the NNMT/CD44v3 axis in regulating HCC metastasis, and presents NNMT as a promising prognostic biomarker and therapeutic target for HCC. Funding Statement: This study was supported by the National Natural Science Foundation of China (No.81672418 and 81871961, to Zhenyu Yin. No. 81802344,to Jie Li. No. 81572335,to Wenxiu Zhao) and the Xiamen Science and Technology Project of People-benefit (No. 3502Z20174089, to J.L.). Declaration of Interests: All of the authors have declared that no competing interests exist. Ethics Approval Statement: Specimen collection was performed after obtaining informed consent from each patient, and the utilization of the tumor materials for research purposes was carried out in accordance with the approved guidelines and approved by the Ethics Committee (No: 20111008) of Zhong Shan Hospital Xiamen University

Advanced Liver Steatosis Accompanies an Increase in the Level of Colonic Pro-inflammatory Bile Acids and Hepatic Inflammation in C57BL/6 Mice Fed a High-fat Diet (P15-027-19)

ObjectivesThe gut-liver axis is implicated in the pathogenesis of liver diseases; however, the mechanistic link between the gut and fatty liver disease severity remains to be characterized. We tested the hypothesis that advanced liver-steatosis accompanies an increase in the concentrations of colonic proinflammatory bile acids and hepatic inflammation in mice fed a high fat (HF) diet. MethodsFour-week-old male C57BL/6 mice (n = 14/group) were fed a HF (45% energy fat) or a low-fat (LF) (10% energy fat) diet for 21 wks. We used biochemical, immunohistological, gas & liquid chromatography with mass spectrometry, RNA sequencing and real-time PCR methods to examine the level of colonic proinflammatory bile acids and hepatic inflammation. ResultsAt the end of the study, body weight and body fat percentage in the HF group were 0.23- and 0.41- fold greater than the LF group, respectively. Compared with the LF group, the HF group exhibited an increase in hepatic lipid droplets, inflammatory cell infiltration, inducible nitric oxide synthase, and hepatocellular ballooning cells (without hepatic Mallory bodies) which are key histological features of advanced hepatic steatosis. Furthermore, nicotinamide n-methyltransferase and selenoprotein P, two hepatic proinflammation-related genes, were upregulated in the HF group. With unique colonic bile acid profiles, the levels of colonic hydrophobic (secondary) bile acids (lithocholic acids and deoxycholic acids) were at least 0.5- fold greater in the HF group compared with the LF group. It is known that these hydrophobic bile acids induce proinflammatory cytokine production (TNF-α and IL-6) in the colon, and exacerbate hepatic inflammation via portal venous circulation. This potential colon-liver axis is consistent with results that the plasma concentrations of TNF-α and IL-6 were 0.5-fold greater in the HF group compared the LF group. ConclusionsOur results demonstrate that advanced liver-steatosis accompanies an increase in colonic proinflammatory bile acid levels and hepatic inflammation, which suggests a cross-talk between colon and liver in mice fed a HF diet. Funding SourcesThis work was funded by the US Department of Agriculture, Agricultural Research Service, CRIS project 3062-51000-050-00D.

Computational de-orphanization of the olive oil biophenol oleacein: Discovery of new metabolic and epigenetic targets

The health promoting effects of extra virgin olive oil (EVOO) relate to its unique repertoire of phenolic compounds. Here, we used a chemoinformatics approach to computationally identify endogenous ligands and assign putative biomolecular targets to oleacein, one of the most abundant secoiridoids in EVOO. Using a structure-based virtual profiling software tool and reference databases containing more than 9000 binding sites protein cavities, we identified 996 putative oleacein targets involving more than 700 proteins. We subsequently identified the high-level functions of oleacein in terms of biomolecular interactions, signaling pathways, and protein-protein interaction (PPI) networks. Delineation of the oleacein target landscape revealed that the most significant modules affected by oleacein were associated with metabolic processes (e.g., glucose and lipid metabolism) and chromatin-modifying enzymatic activities (i.e., histone post-translational modifications). We experimentally confirmed that, in a low-micromolar physiological range (<20 μmol/l), oleacein was capable of inhibiting the catalytic activities of predicted metabolic and epigenetic targets including nicotinamide N-methyltransferase, ATP-citrate lyase, lysine-specific demethylase 6A, and N-methyltransferase 4. Our computational de-orphanization of oleacein provides new mechanisms through which EVOO biophenols might operate as chemical prototypes capable of modulating the biologic machinery of healthy aging.

Nicotinamide N-methyltransferase enhances chemoresistance in breast cancer through SIRT1 protein stabilization

BackgroundNicotinamide N-methyltransferase (NNMT) is overexpressed in various human tumors and involved in the development and progression of several carcinomas. In breast cancer, NNMT was found to be overexpressed in several cell lines. However, the clinical relevance of NNMT in breast cancer is not yet clear.MethodsNNMT expression in breast carcinoma was examined by immunohistochemistry, and then, its relationship with patient clinicopathological characteristics was analyzed. The effects of NNMT on chemoresistance in breast cancer cells were assessed by cell viability, colony formation, and apoptosis assay. The NNMT, SIRT1, p53, and acetyl-p53 proteins, which are involved in NNMT-related chemoresistance, were examined by Western blotting. The SIRT1 mRNA was examined by real-time PCR, and its activity was measured by using the SIRT1 deacetylase fluorometric reagent kit.ResultsNNMT expression was significantly higher (53.9%) in breast carcinoma than in paracancerous tissues (10.0%) and breast hyperplasia (13.3%). A high level of NNMT expression correlated with poor survival and chemotherapy response in breast cancer patients who received chemotherapy. Ectopic overexpression of NNMT significantly inhibited the apoptotic cell death and suppression of colony formation induced by adriamycin and paclitaxel. Mechanistic studies revealed that NNMT overexpression increased SIRT1 expression and promoted its activity. Either inhibition of SIRT1 by EX527 or knockdown of SIRT1 by siRNA could reverse NNMT-mediated resistance to adriamycin and paclitaxel, which suggests that SIRT1 plays a critical role in NNMT-related chemoresistance in breast cancer.ConclusionsThe results of this study demonstrate a novel correlation between the NNMT expression level and patient survival, suggesting that NNMT has the potential to become a new prognostic biomarker to predict the treatment outcomes of the clinical chemotherapy in breast cancer. Moreover, targeting NNMT or downstream SIRT1 may represent a new therapeutic approach to improve the efficacy of breast cancer chemotherapy.

Proteomics reveals NNMT as a master metabolic regulator of cancer-associated fibroblasts.

High grade serous carcinoma (HGSC) has a poor prognosis primarily due to its early dissemination throughout the abdominal cavity. Genomic and proteomic approaches have provided snapshots of the proteogenomics of ovarian cancer (OvCa)1,2, but a systematic examination of both the tumor and stromal compartments is critical to understanding OvCa metastasis. We developed a label-free proteomic workflow to analyze as few as 5,000 formalin-fixed, paraffin embedded cells microdissected from each compartment. The tumor proteome was stable during progression from in situ lesions to metastatic disease; however, the metastasis-associated stroma was characterized by a highly conserved proteomic signature, prominently including the methyltransferase nicotinamide N-methyltransferase (NNMT) and several proteins it regulates. Stromal NNMT expression was necessary and sufficient for functional aspects of the cancer associated fibroblast (CAF) phenotype, including the expression of CAF markers and the secretion of cytokines and oncogenic extracellular matrix. Stromal NNMT expression supported OvCa migration, proliferation, and in vivo growth and metastasis. Expression of NNMT in CAFs led to a depletion of S-adenosyl methionine (SAM) and a reduction in histone methylation associated with widespread gene expression changes in the tumor stroma. This work supports the use of ultra-low input proteomics to identify candidate drivers of disease phenotypes. NNMT is a central, metabolic regulator of CAF differentiation and cancer progression in the stroma that may be therapeutically targeted.

Analysis of nicotinamide N-methyltransferase in oral malignant melanoma and potential prognostic significance.

Oral malignant melanoma (OMM) is an aggressive tumour, and shows deep tissue invasion at initial presentation. The prognosis is worse than that for cutaneous melanoma (CM), and the overall 5-year survival rate is 10-25%. A study of the molecular mechanisms involved in the development of OMM is necessary to identify new prognostic markers. In this study, we evaluated the possible role of nicotinamide N-methyltransferase (NNMT) in OMM. We carried out immunohistochemical analyses to evaluate the expression of NNMT in 15 OMM and 15 CM, measuring the percentage of positive cells and the value of NNMT expression intensity. Furthermore, we explored the relationship between NNMT levels and the prognostic parameters of patients with OMM. NNMT was significantly more expressed in CM compared with OMM, whereas higher staining intensity for NNMT was observed in OMM cases (P<0.05). In addition, a significant relationship was found between NNMT staining intensity and the presence of ulceration (P<0.05). Furthermore, univariate analysis showed a negative effect of NNMT expression on the disease-free survival rate (P<0.05). This study is the first to report the expression of NNMT in OMM and to compare OMM enzyme levels with those detected in CM. Data obtained seem to suggest the presence of potential molecular differences between these two tumours.

Metabolic reprogramming of the stromal epigenome in ovarian cancer metastasis

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

Development of a Suicide Inhibition-Based Protein Labeling Strategy for Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) catalyzes the S-adenosyl-l-methionine-dependent methylation of nicotinamide to form N-methylnicotinamide. This enzyme detoxifies xenobiotics and regulates NAD+ biosynthesis. Additionally, NNMT is overexpressed in various cancers. Herein, we describe the first NNMT-targeted suicide substrates. These compounds, which include 4-chloropyridine and 4-chloronicotinamide, exploit the broad substrate scope of NNMT; methylation of the pyridine nitrogen enhances the electrophilicity of the C4 position, thereby promoting an aromatic nucleophilic substitution by C159, a noncatalytic cysteine. On the basis of this activity, we developed a suicide inhibition-based protein labeling strategy using an alkyne-substituted 4-chloropyridine that selectively labels NNMT in vitro and in cells. In total, this study describes the first NNMT-directed activity-based probes.

Small molecule nicotinamide N-methyltransferase inhibitor activates senescent muscle stem cells and improves regenerative capacity of aged skeletal muscle

Aging is accompanied by progressive declines in skeletal muscle mass and strength and impaired regenerative capacity, predisposing older adults to debilitating age-related muscle deteriorations and severe morbidity. Muscle stem cells (muSCs) that proliferate, differentiate to fusion-competent myoblasts, and facilitate muscle regeneration are increasingly dysfunctional upon aging, impairing muscle recovery after injury. While regulators of muSC activity can offer novel therapeutics to improve recovery and reduce morbidity among aged adults, there are no known muSC regenerative small molecule therapeutics. We recently developed small molecule inhibitors of nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed with aging in skeletal muscles and linked to impairment of the NAD+ salvage pathway, dysregulated sirtuin 1 activity, and increased muSC senescence. We hypothesized that NNMT inhibitor (NNMTi) treatment will rescue age-related deficits in muSC activity to promote superior regeneration post-injury in aging muscle. 24-month old mice were treated with saline (control), and low and high dose NNMTi (5 and 10 mg/kg) for 1-week post-injury, or control and high dose NNMTi for 3-weeks post-injury. All mice underwent an acute muscle injury (barium chloride injection) locally to the tibialis anterior (TA) muscle, and received 5-ethynyl-2′-deoxyuridine systemically to analyze muSC activity. In vivo contractile function measurements were conducted on the injured TA muscle and tissues collected for ex-vivo analyses, including myofiber cross-sectional area (CSA) measurements to assess muscle recovery. Results revealed that muscle stem cell proliferation and subsequent fusion were elevated in NNMTi-treated mice, supporting nearly 2-fold greater CSA and shifts in fiber size distribution to greater proportions of larger sized myofibers and fewer smaller sized fibers in NNMTi-treated mice compared to controls. Prolonged NNMTi treatment post-injury further augmented myofiber regeneration evinced by increasingly larger fiber CSA. Importantly, improved muSC activity translated not only to larger myofibers after injury but also to greater contractile function, with the peak torque of the TA increased by ∼70% in NNMTi-treated mice compared to controls. Similar results were recapitulated in vitro with C2C12 myoblasts, where NNMTi treatment promoted and enhanced myoblast differentiation with supporting changes in the cellular NAD+/NADH redox states. Taken together, these results provide the first clear evidence that NNMT inhibitors constitute a viable pharmacological approach to enhance aged muscle regeneration by rescuing muSC function, supporting the development of NNMTi as novel mechanism-of-action therapeutic to improve skeletal muscle regenerative capacity and functional recovery after musculoskeletal injury in older adults.

PLZF targets developmental enhancers for activation during osteogenic differentiation of human mesenchymal stem cells.

The PLZF transcription factor is essential for osteogenic differentiation of hMSCs; however, its regulation and molecular function during this process is not fully understood. Here, we revealed that the ZBTB16 locus encoding PLZF, is repressed by Polycomb (PcG) and H3K27me3 in naive hMSCs. At the pre-osteoblast stage of differentiation, the locus lost PcG binding and H3K27me3, gained JMJD3 recruitment, and H3K27ac resulting in high expression of PLZF. Subsequently, PLZF was recruited to osteogenic enhancers, influencing H3K27 acetylation and expression of nearby genes important for osteogenic function. Furthermore, we identified a latent enhancer within the ZBTB16/PLZF locus itself that became active, gained PLZF, p300 and Mediator binding and looped to the promoter of the nicotinamide N-methyltransferase (NNMT) gene. The increased expression of NNMT correlated with a decline in SAM levels, which is dependent on PLZF and is required for osteogenic differentiation.

New markers for odontogenic lesions: role of the Nicotinamide N-methyltransferase

New markers for odontogenic lesions: role of the Nicotinamide N-methyltransferase

NNMT Regulates Cancer Immunity and Is a Potential Prognostic Biomarker in Gastric and Colorectal Cancers

Background: Nicotinamide N-methyltransferase (NNMT), a cytosolic enzyme that catalyzes the N-methylation of nicotinamide, has been shown to be critical for promoting cancer progression, but little is known its role in gastric and colorectal cancers. Methods: We explored the role of NNMT in predicting prognosis and immune infiltration of gastric and colorectal cancers. Data was obtained from RNAseq Atlas, Oncomine, tumor Immune Estimation Resource (TIMER) databases, Kaplan-Meier plotter, PrognoScan database and Gene Expression Profiling Interactive Analysis (GEPIA). Findings: NNMT is overexpressed in gastric and colorectal cancers compared to adjacent normal tissues. High NNMT expression was significantly associated with worse outcome in gastric and colorectal cancer patients. However, high expression of NNMT was highly predictive of effectual 5-fluorouracil (5-FU) treatment in HER2 negative gastric cancer patients. Furthermore, there were significant positive correlations between NNMT expression and immune cells infiltration in gastric and colorectal cancers. Conversely, NNMT did not exert any significant effect on prognosis and immune infiltration in lung cancer. Interpretation: NNMT could be a prognostic factor for predicting clinical outcomes and immune infiltration, as well as a molecular target for the treatment of gastric and colorectal cancers. Funding: This work was supported in part by funding from the National Natural Science Foundation of China (No. 81502598). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: Not required.

Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery.

In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-l-proline, named scleric acid. Subsequent rounds of CRISPR/Cas9-mediated gene deletions afforded a selection of biosynthetic gene mutant strains which led to a plausible biosynthetic pathway for scleric acid assembly. Synthetic standards of scleric acid and a key biosynthetic intermediate were also prepared to confirm the chemical structures we proposed. The assembly of scleric acid involves two unique condensation reactions catalysed by a single NRPS module and an ATP-grasp enzyme that link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate, respectively. Scleric acid was shown to exhibit moderate inhibition activity against Mycobacterium tuberculosis, as well as inhibition of the cancer-associated metabolic enzyme nicotinamide N-methyltransferase (NNMT).

Genetic Nicotinamide N-Methyltransferase (Nnmt) Deficiency in Male Mice Improves Insulin Sensitivity in Diet-Induced Obesity but Does Not Affect Glucose Tolerance.

Antisense oligonucleotide knockdown (ASO-KD) of nicotinamide N-methyltransferase (NNMT) in high-fat diet (HFD)-fed mice has been reported to reduce weight gain and plasma insulin levels and to improve glucose tolerance. Using NNMT-ASO-KD or NNMT knockout mice (NNMT-/-), we tested the hypothesis that Nnmt deletion protects against diet-induced obesity and its metabolic consequences in males and females on obesity-inducing diets. We also examined samples from a human weight reduction (WR) study for adipose NNMT (aNNMT) expression and plasma 1-methylnicotinamide (MNAM) levels. In Western diet (WD)-fed female mice, NNMT-ASO-KD reduced body weight, fat mass, and insulin level and improved glucose tolerance. Although NNMT-/- mice fed a standard diet had no obvious phenotype, NNMT-/- males fed an HFD showed strongly improved insulin sensitivity (IS). Furthermore, NNMT-/- females fed a WD showed reduced weight gain, less fat, and lower insulin levels. However, no improved glucose tolerance was observed in NNMT-/- mice. Although NNMT expression in human fat biopsy samples increased during WR, corresponding plasma MNAM levels significantly declined, suggesting that other mechanisms besides aNNMT expression modulate circulating MNAM levels during WR. In summary, upon NNMT deletion or knockdown in males and females fed different obesity-inducing diets, we observed sex- and diet-specific differences in body composition, weight, and glucose tolerance and estimates of IS.