Methionine Restriction Research Articles

Methionine Metabolism – A Gateway to Oral Cancer Epigenetics

Abstract One of the seminal hallmarks of cancer includes nonmutational epigenetic reprogramming. Epigenetic changes are reversible unlike genetic changes which are irreversible and thus hold tremendous potential to be explored for better practical outcome. DNA methylation is the most commonly studied epigenetic alteration. Methionine pathway is linked to DNA methylation. Cancer cells for their metabolism require increased uptake of amino acid and methionine. However, the exact mechanism of DNA methylation in cancer in association to methionine pathway still remains enigmatic. Oral cancer is most commonly associated to smokeless tobacco use. There is sufficient evidence that nitrosamines in tobacco alter the methionine pathway and thus induce aberrant methylation. Moreover, methionine restriction is an emerging strategy to target tumor metabolism. Thus, it makes it all more imperative to understand the basic mechanism operating at the molecular level. This article is an attempt to hypothesis and proposes the possible fundamental changes occurring in the methionine pathway pertaining to oral cancer epigenetics.

Methionine Restriction Impairs Degradation of a Protein that Aberrantly Engages the Endoplasmic Reticulum Translocon.

Proteins that persistently engage endoplasmic reticulum (ER) translocons are degraded by multiple translocon quality control (TQC) mechanisms. In Saccharomyces cerevisiae , the model translocon-associated protein Deg1 -Sec62 is subject to ER-associated degradation (ERAD) by the Hrd1 ubiquitin ligase and, to a lesser extent, proteolysis mediated by the Ste24 protease. In a recent screen, we identified nine methionine-biosynthetic genes as candidate TQC regulators. Here, we found methionine restriction impairs Hrd1-independent Deg1 -Sec62 degradation. Beyond revealing methionine as a novel regulator of TQC, our results urge caution when working with laboratory yeast strains with auxotrophic mutations, often presumed not to influence cellular processes under investigation.

Effects of methionine intake on cognitive function in mild cognitive impairment patients and APP/PS1 Alzheimer's Disease model mice: Role of the cystathionine-β-synthase/H2S pathway

As a dietary intervention, methionine restriction (MR) has been reported to increase longevity and improve metabolism disorders. However, the effects of MR on alleviating neurodegenerative diseases such as Alzheimer's disease (AD) are largely unexplored. Here we sought to investigate the neuroprotective effects of low methionine intake in mild cognitive impairment (MCI) patients and APP/PS1 AD model mice, and to uncover the underlying mechanisms. In a cohort composed of 45 individuals diagnosed with MCI and 61 healthy controls without cognitive impairment, methionine intake was found to be positively associated with the increased risk of MCI, where no sex differences were observed. We further conducted a 16-week MR intervention (0.17% methionine, w/w) on APP/PS1 AD model mice. Although MR reduced Aβ accumulation in the brain of both male and female APP/PS1 mice, MR improved cognitive function only in male mice, as assessed by the Morris water maze test. Consistently, MR restored synapse ultrastructure and alleviated mitochondrial dysfunction by enhancing mitochondrial biogenesis in the brain of male APP/PS1 mice. Importantly, MR effectively balanced the redox status and activated cystathionine-β-synthase (CBS)/H2S pathway in the brain of male APP/PS1 mice. Together, our study indicated that lower dietary methionine intake is associated with improved cognitive function, in which CBS/H2S pathway plays an essential role. MR could be a promising nutritional intervention for preventing AD development.

Sexual dimorphism in the response to dietary restriction in mice: A systematic review of the literature

Background: Dietary restriction (DR) is a widely used experimental intervention in aging research due to its consistent ability to extend lifespan in most species tested. DR is an all-encompassing term describing interventions that restrict some aspect of nutrition - from calorie amount to calorie type to timing of food intake - and yet share common functional endpoints including extended longevity, but also improvements in healthspan, or the time spent in good health, as well as metabolic fitness and stress resistance. Recent studies highlight the preponderance of sexual dimorphisms in the response to DR and argue for the importance of inclusion of both sexes in preclinical research. OBJECTIVE: We set out to perform a comprehensive assessment of documented health and lifespan outcomes of interventional DR studies in mice that display sexual dimorphism. METHODS: A systematic literature search was conducted according to the PRISMA statement to identify mouse DR studies in which both sexes were included using PubMed. The specific DR interventions examined included calorie restriction (CR), intermittent fasting (IF), protein restriction (PR) and methionine restriction (MetR), with experimental endpoints focused on lifespan and healthspan. RESULTS: Sexual dimorphism in the lifespan and healthspan effects of various DR regimens is a common finding in mice, with the magnitude and direction of dimorphic responses influenced by the specific dietary intervention as well as the strain of mouse used in the study. CONCLUSIONS: Despite the fact that preclinical lifespan and healthspan analyses in mice reveal sexual dimorphism in the response to DR, there is still a large gap in our understanding of how sex affects dietary outcomes. More preclinical research comparing both sexes in the same study with better attention to reporting metrics during peer review and in easily searchable text including title and abstract is required to further our understanding of the impact of sex on health and lifespan in response to DR in rodent studies.

Methionine-Restricted Diet: A Feasible Strategy Against Chronic or Aging-Related Diseases

Dietary methionine restriction (MR) has been associated with multifaceted health-promoting effects. MR is conducive to prevention of several chronic diseases and cancer, and extension of lifespan. A growing number of studies on new phenotypes and mechanisms of MR have become available in the past five years, especially in angiogenesis, neurodegenerative diseases, intestinal microbiota, and intestinal barrier function. In this review, we summarize the characteristics and advantages of MR, and current knowledge on the physiological responses and effects of MR on chronic diseases and aging-associated pathologies. Potential mechanisms, in which hydrogen sulfide, fibroblast growth factor 21, gut microbiota, short-chain fatty acids, and so on are involved, are discussed. Moreover, directions for epigenetics and gut microbiota in an MR diet are presented in future perspectives. This review comprehensively summarizes the novel roles and interpretations of the mechanisms underlying MR in the prevention of chronic diseases and aging.

Synergy of oral recombinant methioninase (rMETase) and 5-fluorouracil on poorly differentiated gastric cancer

Gastric cancer is highly malignant and recalcitrant to first line chemotherapies that include 5-fluorouracil (5-FU). Cancer cells are addicted to methionine for their proliferation and survival. Methionine addiction of cancer is known as the Hoffman effect. Methionine restriction with recombinant methioninase (rMETase) has been shown to selectively starve cancer cells and has shown synergy with cytotoxic chemotherapy including 5-FU. The present study aimed to investigate the efficacy of rMETase alone and the combination with 5-FU on poorly differentiated human gastric cancer cell lines (MKN45, NUGC3, and NUGC4) in vitro and vivo. rMETase suppressed the tumor growth of 3 kinds of poorly differentiated gastric cancer cells in vitro. The fluorescence ubiquitination-based cell cycle indicator (FUCCI) demonstrated cancer cells treated with rMETase were selectively trapped in the S/G2 phase of the cell cycle. In the present study, subcutaneous MKN45 gastric cancer models were randomized into four groups when the tumor volume reached 100 mm3: G1: untreated control; G2: 5-FU (i.p., 50 mg/kg, weekly, three weeks); G3: oral-rMETase (o-rMETase) (p.o., 100 units/body, daily, three weeks); G4: 5-FU with o-rMETase (5-FU; i.p., 50 mg/kg, weekly, three weeks o-rMETase; p.o., 100 units/body, daily, three weeks). All mice were sacrificed on day 22. Body weight and estimated tumor volume were measured twice a week. 5-FU and o-rMETase suppressed tumor growth as monotherapies on day 18 (p = 0.044 and p = 0.044). However, 5-FU combined with o-rMETase was significantly superior to each monotherapy (p < 0.001 and p < 0.001, respectively) and induced extensive necrosis compared to other groups. The combination of 5-FU and o-rMETase shows promise for transformative therapy for poorly differentiated gastric cancer in the clinic.

Effects of dietary methionine restriction on age-related changes in perivascular and beiging adipose tissues in the mouse.

Perivascular adipose tissue (PVAT) regulates vascular health. Dietary methionine restriction (MetR) impacts age-related adiposity, and this study addresses its effects in PVAT. Male C57BL/6 mice at 8, 52, and 102 weeks of age were fed a standard (0.86%) or low-methionine (0.12%) diet for 52 weeks in 8-week-old and 52-week-old mice and for 15 weeks in 102-week-old mice. Mice with dietary MetR were resistant to weight gain and maintained a healthy blood profile. Aging increased lipid accumulation, and MetR reversed this phenotype. Notch signaling in inguinal white adipose tissue (iWAT) was decreased by MetR but increased in gonadal white adipose tissue. However, the Notch phenotype of brown adipose tissue (BAT) was not affected by MetR. Uncoupling protein 1 (UCP1) was increased in PVAT, iWAT, and BAT by MetR when initiated in young mice, but this effect was lost in middle-aged mice. Lipid in mouse PVAT peaked at 1year of age, consistent with peak body mass. MetR reduced body weight, normalized metabolic parameters, and decreased lipid in PVAT in all age cohorts. Mice fed a MetR diet from early maturity to 1 year of age displayed an increased thermogenic adipocyte phenotype in iWAT, PVAT, and BAT, all tissues with thermogenic capacity.

Maternal dietary methionine restriction alters hepatic expression of one-carbon metabolism and epigenetic mechanism genes in the ducklings

BackgroundEmbryonic and fetal development is very susceptible to the availability of nutrients that can interfere with the setting of epigenomes, thus modifying the main metabolic pathways and impacting the health and phenotypes of the future individual. We have previously reported that a 38% reduction of the methyl donor methionine in the diet of 30 female ducks reduced the body weight of their 180 mule ducklings compared to that of 190 ducklings from 30 control females. The maternal methionine-restricted diet also altered plasmatic parameters in 30 of their ducklings when compared to that of 30 ducklings from the control group. Thus, their plasma glucose and triglyceride concentrations were higher while their free fatty acid level and alanine transaminase activity were decreased. Moreover, the hepatic transcript level of 16 genes involved in pathways related to energy metabolism was significantly different between the two groups of ducklings. In the present work, we continued studying the liver of these newly hatched ducklings to explore the impact of the maternal dietary methionine restriction on the hepatic transcript level of 70 genes mostly involved in one-carbon metabolism and epigenetic mechanisms.ResultsAmong the 12 genes (SHMT1, GART, ATIC, FTCD, MSRA, CBS, CTH, AHCYL1, HSBP1, DNMT3, HDAC9 and EZH2) identified as differentially expressed between the two maternal diet groups (p-value < 0.05), 3 of them were involved in epigenetic mechanisms. Ten other studied genes (MTR, GLRX, MTHFR, AHCY, ADK, PRDM2, EEF1A1, ESR1, PLAGL1, and WNT11) tended to be differently expressed (0.05 < p-value < 0.10). Moreover, the maternal dietary methionine restriction altered the number and nature of correlations between expression levels of differential genes for one-carbon metabolism and epigenetic mechanisms, expression levels of differential genes for energy metabolism, and phenotypic traits of ducklings.ConclusionThis avian model showed that the maternal dietary methionine restriction impacted both the mRNA abundance of 22 genes involved in one-carbon metabolism or epigenetic mechanisms and the mRNA abundance of 16 genes involved in energy metabolism in the liver of the newly hatched offspring, in line with the previously observed changes in their phenotypic traits.

Cysteine restriction-specific effects of sulfur amino acid restriction on lipid metabolism.

Decreasing the dietary intake of methionine exerts robust anti-adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti-adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine- and cysteine-titrated diets, we demonstrate that the anti-adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non-glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck-M. In mice, the magnitude of SAAR-induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age-at-onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross-sectional epidemiological study. Controlled feeding of low-SAA, high-polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.

MLL (KMT2A)-rearranged Acute Lymphoblastic Leukemias Are Addicted to S-Adenosyl Methionine (SAM): Implications for Therapy

MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is an aggressive subset associated with poor survival and relapse representing 5% of childhood ALL cases and 75% of infant leukemias. Though current intensive chemotherapy regimens have improved survival in childhood ALL overall, they fail to cure many of these high-risk patient subgroups and inflict significant long-term side effects, demonstrating the urgent need for novel therapies. While exploring the effect of different amino acid depletion strategies, we observed rapid induction of apoptosis upon methionine restriction (MR) in MLLr cells. The histone methyltransferase function of the MLL fusion protein complex requires increased amounts of the methionine metabolite s-adenosylmethionine (SAM) to maintain its hypermethylated state, creating a selective sensitivity of MLLr ALL for perturbations in methionine availability. To explore how MR impinges on MLLr cells' metabolic state we performed global metabolomics on SEM cells and observed rapid effects on metabolism, with 416 metabolites out of the 650 measured differing significantly upon 12 hours of MR. We performed in parallel mRNA sequencing on SEM cells to understand the effect of MR on gene expression and identified 1351 differentially expressed genes. Integrated transcriptomics and metabolomics analysis revealed several key pathways interlinked with SAM, including the folate cycle, polyamine synthesis, arginine/redox metabolism, nucleotide synthesis, and the creatine/urea cycle, with several SAM-dependent enzymes and key byproducts in these pathways being significantly reduced upon MR. Moreover, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells, confirming the critical role of SAM in this leukemia subset. To better understand the effect of methionine and SAM restriction on epigenetic modifications, we looked at histone methylation in a panel of MLLr and non-MLLr B-ALL cell lines and observed a global suppression of methylation upon MR specifically in the pediatric MLLr cells. Additionally, gene expression analysis revealed downregulation of key MLL fusion target genes associated with proliferation and maintenance of the oncogenic phenotype in response to MR, including ZMYND8, ERG, BCL2, PROM1, HMX3, HOXA10, HMGA2, and PBX3. Several of these gene targets are known to be transcriptionally upregulated in MLLr leukemia via aberrant H3K79 methylation. Consistent with this notion, knockdown of the H3K79 demethylase, KDM2B, displayed a more MR-resistant phenotype than its wildtype counterparts. Taken together, MR has a direct effect on global histone methylation in MLLr ALL cells, which in turn disturbs the MLL gene signature critical for their survival. Lastly, immunocompromised mice were engrafted with MLLr SEM cells and treated with a 95% MR diet while leukemia progression was monitored by flow cytometry. The MR diet was well tolerated with minimal weight loss, methionine levels in plasma were roughly 50% reduced compared to controls, and that alone was enough to significantly delay leukemic growth (p = 0.015). While several compounds targeting MAT2A, the enzyme that produces SAM from methionine, are currently in Phase 1 clinical trials, the availability of an FDA approved methionine-free formula for infants allows for rapid translation to the clinic. As a follow-up, we also performed a drug screen using 214 compounds to identify drugs that interact with MR, showing that vinca-alkaloids, including vincristine, strongly synergize. Validation experiments demonstrate that MR triggers a stress induced G2 arrest, priming cells for mitotic spindle destruction with vincristine. In vivo experiments are currently underway using patient-derived xenografts exploring synergistic potential of vincristine with targeting methionine metabolism. In summary, we found that MLLr leukemic cells are selectively vulnerable to perturbations of the methionine cycle due to their increased need for SAM to maintain hypermethylation and aberrant gene activation. Limiting methionine and/or SAM availability provides an attractive adjuvant therapy potentiating the effects of current or novel (targeted) therapies for MLLr leukemia.

Stemness of Normal and Cancer Cells: The Influence of Methionine Needs and SIRT1/PGC-1α/PPAR-α Players.

Stem cells are a population of undifferentiated cells with self-renewal and differentiation capacities. Normal and cancer stem cells share similar characteristics in relation to their stemness properties. One-carbon metabolism (OCM), a network of interconnected reactions, plays an important role in this dependence through its role in the endogenous synthesis of methionine and S-adenosylmethionine (SAM), the universal donor of methyl groups in eukaryotic cells. OCM genes are differentially expressed in stem cells, compared to their differentiated counterparts. Furthermore, cultivating stem cells in methionine-restricted conditions hinders their stemness capacities through decreased SAM levels with a subsequent decrease in histone methylation, notably H3K4me3, with a decrease in stem cell markers. Stem cells' reliance on methionine is linked to several mechanisms, including high methionine flux or low endogenous methionine biosynthesis. In this review, we provide an overview of the recent discoveries concerning this metabolic dependence and we discuss the mechanisms behind them. We highlight the influence of SIRT1 on SAM synthesis and suggest a role of PGC-1α/PPAR-α in impaired stemness produced by methionine deprivation. In addition, we discuss the potential interest of methionine restriction in regenerative medicine and cancer treatment.

On the illusion of auxotrophy: met15Δ yeast cells can grow on inorganic sulfur, thanks to the previously uncharacterized homocysteine synthase Yll058w

Organisms must either synthesize or assimilate essential organic compounds to survive. The homocysteine synthase Met15 has been considered essential for inorganic sulfur assimilation in yeast since its discovery in the 1970s. As a result, MET15 has served as a genetic marker for hundreds of experiments that play a foundational role in eukaryote genetics and systems biology. Nevertheless, we demonstrate here through structural and evolutionary modeling, invitro kinetic assays, and genetic complementation, that an alternative homocysteine synthase encoded by the previously uncharacterized gene YLL058W enables cells lacking Met15 to assimilate enough inorganic sulfur for survival and proliferation. These cells however fail to grow in patches or liquid cultures unless provided with exogenous methionine or other organosulfurs. We show that this growth failure, which has historically justified the status of MET15 as a classic auxotrophic marker, is largely explained by toxic accumulation of the gas hydrogen sulfide because of a metabolic bottleneck. When patched or cultured with a hydrogen sulfide chelator, and when propagated as colony grids, cells without Met15 assimilate inorganic sulfur and grow, and cells with Met15 achieve even higher yields. Thus, Met15 is not essential for inorganic sulfur assimilation in yeast. Instead, MET15 is the first example of a yeast gene whose loss conditionally prevents growth in a manner that depends on local gas exchange. Our results have broad implications for investigations of sulfur metabolism, including studies of stress response, methionine restriction, and aging. More generally, our findings illustrate how unappreciated experimental variables can obfuscate biological discovery.

MTORC1-c-Myc pathway rewires methionine metabolism for HCC progression through suppressing SIRT4 mediated ADP ribosylation of MAT2A

BackgroundExploiting cancer metabolism during nutrient availability holds immense potential for the clinical and therapeutic benefits of hepatocellular carcinoma (HCC) patients. Dietary methionine is a metabolic dependence of cancer development, but how the signal transduction integrates methionine status to achieve the physiological demand of cancer cells remains unknown.MethodsLow or high levels of dietary methionine was fed to mouse models with patient-derived xenograft or diethyl-nitrosamine induced liver cancer. RNA sequence and metabolomics were performed to reveal the profound effect of methionine restriction on gene expression and metabolite changes. Immunostaining, sphere formation assays, in vivo tumourigenicity, migration and self-renewal ability were conducted to demonstrate the efficacy of methionine restriction and sorafenib.ResultsWe discovered that mTORC1-c-Myc-SIRT4 axis was abnormally regulated in a methionine-dependent manner and affected the HCC progression. c-Myc rewires methionine metabolism through TRIM32 mediated degradation of SIRT4, which regulates MAT2A activity by ADP-ribosylation on amino acid residue glutamic acid 111. MAT2A is a key enzyme to generate S-adenosylmethionine (SAM). Loss of SIRT4 activates MAT2A, thereby increasing SAM level and dynamically regulating gene expression, which triggers the high proliferation rate of tumour cells. SIRT4 exerts its tumour suppressive function with targeted therapy (sorafenib) by affecting methionine, redox and nucleotide metabolism.ConclusionsThese findings establish a novel characterization of the signaling transduction and the metabolic consequences of dietary methionine restriction in malignant liver tissue of mice. mTORC1, c-Myc, SIRT4 and ADP ribosylation site of MAT2A are promising clinical and therapeutic targets for the HCC treatment.

Abstract 10708: Dietary Methionine Restriction Protects Against Abdominal Aortic Aneurysm via Amino Acid Starvation Response

Introduction: Dietary intervention emerges as a promising non-medicinal avenue for the treatment of a wide range of diseases. Limiting the intake of single amino acid has been shown to produce health benefits similar to that of calorie restriction yet without significant risks of malnutrition. One of the established regimen, methionine restriction (MR), was recently shown to confer pleiotropic benefits in cardiometabolic disease models, largely attributed to the activation of the GCN2-mediated amino acid starvation response pathway. However, the therapeutic and mechanistic implications of dietary amino acid restriction remain unknown in abdominal aortic aneurysm (AAA), a lethal vascular disease that is without a cure other than the traumatic surgical repairs. Herein, we sought to probe the outcome of MR as well as the role of GCN2 in AAA. Methods and Results: Following a 2-week pre-treatment with MR (20% methionine level) or iso-caloric control diet, male Sprague Dawley rats were subjected to intraluminal elastase perfusion. At day 7, animals in the MR group showed significantly less aneurysmal dilation in comparison to control. To investigate the contribution from the GCN2-mediated amino acid starvation response, we subjected animals to the same dietary regimen of MR or control, followed by GCN2 silencing in the elastase-challenged aortic segments through localized infusion with GCN2-targeting or scrambled shRNA lentivirus. Indeed, tuning down aortic GCN2 effectively compromised the benefits exerted by MR against AAA. Finally, we determined whether GCN2 constitutes an intrinsic protector against aortic degeneration using GCN2 knockout mice ( Gcn2 -/- ). Following periaortic elastase challenge and β-aminopropionitrile treatment, Gcn2 -/- mice displayed exacerbated expansion of aneurysmal lesions in comparison to Gcn2 +/+ control mice at day 28 post procedure. Conclusions: Our study provides the first experimental evidence supporting a potential role of dietary amino acid restriction (e.g., MR) in the prevention of AAA development. Additionally, GCN2-mediated amino acid starvation response possibly underlies the anti-AAA benefit exerted by MR and serves as an intrinsic protector against AAA pathogenesis.

Methionine Restriction Improves Cognitive Ability by Alleviating Hippocampal Neuronal Apoptosis through H19 in Middle-Aged Insulin-Resistant Mice.

LncRNA H19 has been reported to regulate apoptosis and neurological diseases. Hippocampal neuron apoptosis damages cognitive ability. Methionine restriction (MR) can improve cognitive impairment. However, the effect of MR on hippocampal neuronal apoptosis induced by a high-fat diet (HFD) in middle-aged mice remains unclear. For 25 weeks, middle-aged mice (C57BL/6J) were given a control diet (CON, 0.86% methionine + 4.2% fat), a high-fat diet (HFD, 0.86% methionine + 24% fat), or an HFD + MR diet (HFMR, 0.17% methionine + 24% fat). The HT22 cells were used to establish the early apoptosis model induced by high glucose (HG). In vitro, the results showed that MR significantly improved cell viability, suppressed the generation of ROS, and rescued HT22 cell apoptosis in a gradient-dependent manner. In Vivo, MR inhibited the damage and apoptosis of hippocampal neurons caused by a high-fat diet, reduced hippocampal oxidative stress, improved hippocampal glucose metabolism, relieved insulin resistance, and enhanced cognitive ability. Furthermore, MR could inhibit the overexpression of H19 and caspase-3 induced by HFD, HG, or H2O2 in vivo and in vitro, and promoted let-7a, b, e expression. These results indicate that MR can protect neurons from HFD-, HG-, or H2O2-induced injury and apoptosis by inhibiting H19.

Oral-recombinant Methioninase in Combination With Rapamycin Eradicates Osteosarcoma of the Breast in a Patient-derived Orthotopic Xenograft Mouse Model

Primary osteosarcoma of the breast is a very rare malignancy that shares histological features with osteosarcoma. It is also highly sensitive to methionine restriction due to methionine addiction. We previously established a patient-derived orthotopic xenograft (PDOX) nude-mouse model derived from tumor tissue of a patient with primary mammary osteosarcoma. In the present study, we investigated the efficacy of oral-recombinant methioninase (o-rMETase), combined with rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on a mammary osteosarcoma PDOX nude-mouse model. The PDOX mouse model was established by surgically transplanting a specimen of primary osteosarcoma of the breast into the mammary gland of nude mice. Mice implanted with tumors were randomly divided into four groups: Control group, N=5; rapamycin-treated group, N=5; o-rMETase-treated group, N=5; and a group treated with the combination of o-rMETase and rapamycin, N=5. Mice were treated for 2 weeks after transplantation, and tumor volume was measured during the treatment period. Treatment with the combination of rapamycin and o-rMETase eradicated the osteosarcoma of the breast compared to the untreated control (p=0.000008). o-rMETase alone did not significantly inhibit tumor growth, and rapamycin alone only partially inhibited the tumor (p=0.78 and p=0.018, respectively) compared to the untreated control. There was not a significant difference in mouse weight between the groups. The combination of rapamycin and o-rMETase was highly effective against primary osteosarcoma of the breast in a PDOX model, suggesting a future clinical strategy for this rare cancer type that currently has no first-line treatment.

Methionine restriction forces Epstein-Barr virus out of latency

EBV gene expression is repressed during viral latency to prevent an immune response, but it is not known how metabolism contributes to this silencing. In this issue of Cell Metabolism, Guo etal. describe how methionine restriction reactivates the expression of EBV genes, offering new therapeutic approaches against EBV-driven diseases.

Methionine metabolism controls the B cell EBV epigenome and viral latency

Epstein-Barr virus (EBV) subverts host epigenetic pathways to switch between viral latency programs, colonize the B cell compartment, and reactivate. Within memory B cells, the reservoir for lifelong infection, EBV genomic DNA and histone methylation marks restrict gene expression. But this epigenetic strategy also enables EBV-infected tumors, including Burkitt lymphomas, to evade immune detection. Little is known about host cell metabolic pathways that support EBV epigenome landscapes. We therefore used amino acid restriction, metabolomic, and CRISPR approaches to identify that an abundant methionine supply and interconnecting methionine and folate cycles maintain Burkitt EBV gene silencing. Methionine restriction, or methionine cycle perturbation, hypomethylated EBV genomes and de-repressed latent membrane protein and lytic gene expression. Methionine metabolism also shaped EBV latency gene regulation required for B cell immortalization. Dietary methionine restriction altered murine Burkitt xenograft metabolomes and de-repressed EBV immunogens invivo. These results highlight epigenetic/immunometabolism crosstalk supporting the EBV B cell life cycle and suggest therapeutic approaches.

Mass Spectrometric Metabolic Fingerprinting of 2-Deoxy-D-Glucose (2-DG)-Induced Inhibition of Glycolysis and Comparative Analysis of Methionine Restriction versus Glucose Restriction under Perfusion Culture in the Murine L929 Model System.

All forms of restriction, from caloric to amino acid to glucose restriction, have been established in recent years as therapeutic options for various diseases, including cancer. However, usually there is no direct comparison between the different restriction forms. Additionally, many cell culture experiments take place under static conditions. In this work, we used a closed perfusion culture in murine L929 cells over a period of 7 days to compare methionine restriction (MetR) and glucose restriction (LowCarb) in the same system and analysed the metabolome by liquid chromatography mass spectrometry (LC-MS). In addition, we analysed the inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) over a period of 72 h. 2-DG induced very fast a low-energy situation by a reduced glycolysis metabolite flow rate resulting in pyruvate, lactate, and ATP depletion. Under perfusion culture, both MetR and LowCarb were established on the metabolic level. Interestingly, over the period of 7 days, the metabolome of MetR and LowCarb showed more similarities than differences. This leads to the conclusion that the conditioned medium, in addition to the different restriction forms, substantially reprogramm the cells on the metabolic level.

α‐Ketobutyrate Production under Continuous‐Flow Conditions Catalyzed by Immobilized L‐Methionine γ‐Lyase

Abstractl‐Methionine γ‐lyase (MGL) is a PLP‐dependent enzyme which catalyzes the α, γ‐elimination reaction in sulfur containing amino acids, such as L‐methionine and L‐cysteine. Its major applications are on metabolic restriction of methionine to cancer cells, food industry (cheese flavor) and for the synthesis of d and l‐homoalanine. Herein, we present the immobilization and use of this new immobilized biocatalyst under batch and continuous‐flow conditions. The best optimized continuous‐flow condition allowed 89 % conversion in 30 min of residence time with good recyclability of the biocatalyst.