P13.07 Cysteine induces glioblastoma cytotoxicity through mitochondrial reductive stress

BACKGROUND Glioblastoma (GBM) remains a poorly treatable disease with high mortality. Tumor metabolism in GBM is a critical mechanism responsible for accelerated growth because of upregulation of glucose, amino acid, and fatty acid utilization. However, therapies targeting GBM metabolism, whether through the use of small-molecule compounds or dietary interventions to limit nutrient sources, have failed in clinical trials. Metabolic bypass is an important mechanism that is often overlooked in GBM trials, since many trials have focused instead on combining anti-metabolic therapy with cytotoxic treatments. The goal of this research is to use a multi-pronged treatment approach with targeted drug and dietary therapy to leverage metabolic susceptibilities in GBM. MATERIALS AND METHODS We first interrogated the TCGA database and a cancer metabolite database for alterations in glucose and amino acid signatures in GBM relative to other human cancers and relative to low-grade glioma. We identified the amino acid cysteine as contributing to a novel metabolic susceptibility pathway in GBM. To study the role of cysteine in GBM pathogenesis, we treated patient-derived GBM cells with a variety of FDA-approved cysteine-promoting compounds in vitro, including N-acetylcysteine (NAC). We measured cell proliferation, energy production, mitochondrial metabolism, and reactive oxygen species to study mechanisms of oxidoreductive stress. Results: From our TCGA and cancer metabolite database analyses, we found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers and that GBM exhibits high levels of cysteine-related metabolites compared to low-grade gliomas. Cysteine compounds, including NAC, reduce growth of GBM cells, which is exacerbated by glucose deprivation. This growth inhibition is associated with reduced mitochondrial metabolism, manifest by reduction in ATP generation, NADPH/NADP+ ratio, mitochondrial membrane potential, and oxygen consumption rate. Through measurement of mitochondrial hydrogen peroxide, we found that NAC-treated cells exhibit a paradoxical increase in mitochondrial hydrogen peroxide levels, likely due to inhibition of thioreductase and glutathione reductase systems. Through genetic and pharmacological studies, we found that induction of thioredoxin-2 rescues NAC-mediated cytotoxicity and that inhibition of thioreductase and glutathione reductase exacerbates mitochondrial toxicity and reductive stress. CONCLUSIONS We show that cysteine compounds reduce cell growth and induce mitochondrial toxicity in GBM through reductive stress. This metabolic phenotype is exacerbated by glucose deprivation. This pathway is targetable with FDA-approved cysteine-promoting compounds and could synergize with glucose-lowering treatments, including the ketogenic diet, for GBM.

Metabolic Characterisation of Magnetospirillum Gryphiswaldense MSR-1 Using LC-MS-Based Metabolite Profiling

<p>Magnetosomes are nano-sized magnetic nanoparticles with exquisite properties that can be used in a wide range of healthcare and biotechnological applications. They are biosynthesised by magnetotactic bacteria (MTB) such as <i>Magnetospirillum gryphiswaldense </i>MSR-1 (<i>Mgryph</i>). However, magnetosome bioprocessing yields low quantities compared to chemical synthesis of magnetic nanoparticles. Therefore, the understanding of the intracellular metabolites and the metabolic networks related to <i>Mgryph</i> growth and magnetosome formation are vital to unlock the potential of this organism to develop improved bioprocesses. In this work, we investigated the metabolism of <i>Mgryph</i> using untargeted metabolomics. Liquid chromatography-mass spectrometry (LC-MS) was performed to profile spent medium samples of <i>Mgryph </i>cells grown under O₂-limited (n=6) and O₂-rich conditions (n=6) corresponding to magnetosome- and non-magnetosome producing cells, respectively. Cross-validated multivariate, univariate and pathway enrichment analyses were conducted to identify significantly altered metabolites and pathways. Rigorous metabolite identification was carried out using authentic standards, <i>Mgryph-</i>specific metabolite database<i> </i>and<i> </i>MS/MS mzCloud database. PCA and OPLS-DA showed clear separation and clustering of sample groups with cross-validation values of R²X=0.76, R²Y=0.99 and Q²=0.98 in OPLS-DA. As a result, 50 metabolites linked to 45 metabolic pathways were found significantly altered in the tested conditions including glycine, serine and threonine; butanoate; alanine, aspartate and glutamate metabolism; aminoacyl-tRNA biosynthesis and; pyruvate and citric acid cycle (TCA) metabolisms. Our findings demonstrate the potential of LC-MS to characterise key metabolites in <i>Mgryph</i> and will contribute to further understand the metabolic mechanisms that affect <i>Mgryph</i> growth and magnetosome formation. <i></i></p>

Metabolic Characterisation of Magnetospirillum Gryphiswaldense MSR-1 Using LC-MS-Based Metabolite Profiling

<p>Magnetosomes are nano-sized magnetic nanoparticles with exquisite properties that can be used in a wide range of healthcare and biotechnological applications. They are biosynthesised by magnetotactic bacteria (MTB) such as <i>Magnetospirillum gryphiswaldense </i>MSR-1 (<i>Mgryph</i>). However, magnetosome bioprocessing yields low quantities compared to chemical synthesis of magnetic nanoparticles. Therefore, the understanding of the intracellular metabolites and the metabolic networks related to <i>Mgryph</i> growth and magnetosome formation are vital to unlock the potential of this organism to develop improved bioprocesses. In this work, we investigated the metabolism of <i>Mgryph</i> using untargeted metabolomics. Liquid chromatography-mass spectrometry (LC-MS) was performed to profile spent medium samples of <i>Mgryph </i>cells grown under O₂-limited (n=6) and O₂-rich conditions (n=6) corresponding to magnetosome- and non-magnetosome producing cells, respectively. Cross-validated multivariate, univariate and pathway enrichment analyses were conducted to identify significantly altered metabolites and pathways. Rigorous metabolite identification was carried out using authentic standards, <i>Mgryph-</i>specific metabolite database<i> </i>and<i> </i>MS/MS mzCloud database. PCA and OPLS-DA showed clear separation and clustering of sample groups with cross-validation values of R²X=0.76, R²Y=0.99 and Q²=0.98 in OPLS-DA. As a result, 50 metabolites linked to 45 metabolic pathways were found significantly altered in the tested conditions including glycine, serine and threonine; butanoate; alanine, aspartate and glutamate metabolism; aminoacyl-tRNA biosynthesis and; pyruvate and citric acid cycle (TCA) metabolisms. Our findings demonstrate the potential of LC-MS to characterise key metabolites in <i>Mgryph</i> and will contribute to further understand the metabolic mechanisms that affect <i>Mgryph</i> growth and magnetosome formation. <i></i></p>

Reprogramming of the apoplast metabolome of Lolium perenne upon infection with the mutualistic symbiont Epichloë festucae

SummaryEpichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-house Epichloë metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

DIMEdb: an integrated database and web service for metabolite identification in direct infusion mass spectrometery

MotivationMetabolomics involves the characterisation, identification, and quantification of small molecules (metabolites) that act as the reaction intermediates of biological processes. Over the past few years, we have seen wide scale improvements in data processing, database, and statistical analysis tools. Direct infusion mass spectrometery (DIMS) is a widely used platform that is able to produce a global fingerprint of the metabolome, without the requirement of a prior chromatographic step - making it ideal for wide scale high-throughput metabolomics analysis. In spite of these developments, metabolite identification still remains a key bottleneck in untargeted mass spectrometry-based metabolomics studies. The first step of the metabolite identification task is to query masses against a metaboite database to get putative metabolite annotations. Each existing metabolite database differs in a number of aspects including coverage, format, and accessibility - often limiting the user to a rudimentary web interface. Manually combining multiple search results for a single experiment where there may be potentially hundreds of masses to investigate becomes an incredibly arduous task.ResultsTo facilitate unified access to metabolite information we have created the Direct Infusion MEtabolite database (DIMEdb), a comprehensive web-based metabolite database that contains over 80,000 metabolites sourced from a number of renowned metabolite databases of which can be utilised in the analysis and annotation of DIMS data. To demostrate the efficacy of DIMEdb, a simple use case for metabolic identification is presented. DIMEdb aims to provide a single point of access to metabolite information, and hopefully facilitate the development of much needed bioinformatic tools.AvailabilityDIMEdb is freely available at https://[email protected] informationSupplementary data are available at Bioinformatics online.

Systematic isolation of microbial metabolites for natural products depository (NPDepo)

A microbial fraction library has been constructed as a part of RIKEN Natural Products Depository (NPDepo) to discover and isolate novel metabolites with unique biological activity from microbial sources efficiently and rapidly. The fraction library was made by a systematic separation method based on basic chromatographic techniques. Each fraction in the library was analyzed by liquid chromatography/mass spectrometry (LC/MS) to reveal physicochemical properties of each metabolite within the fraction, and the results were applied to construct a database for rapid discovery of novel and structurally unique compounds. We developed a new type of metabolite database called MP (microbial products) plot to visualize each metabolite on a 2D area. The combination of the fraction library and the database led to the discovery and isolation of novel metabolites, verticilactam, which was a 16-membered macrolactam with an unprecedented β-keto-amide moiety, and spirotoamides A and B, which had a highly substituted 6,6-spiroaccetal moiety and a carboxamide moiety. Moreover, based on the utilization of the MP plot, the new capacity of a streptomycete to produce specific metabolites was discovered.