Dietary methionine starvation impairs acute myeloid leukemia progression.

Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, nuclear magnetic resonance-based metabolite quantifications and 13C-tracing, polysomal profiling, and chromatin immunoprecipitation sequencing, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis, and induced a cell cycle block. Reactive oxygen species levels were not increased following methionine depletion, and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.

The glutamate/aspartate transporter EAAT1 is crucial for T-cell acute lymphoblastic leukemia proliferation and survival.

T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients have refractory disease or relapse and die from the disease. To improve patient outcome new therapeutics are needed. With the aim to identify new therapeutic targets, we combined the analysis of T-ALL gene expression and metabolism to identify the metabolic adaptations that T-ALL cells exhibit. We found that glutamine uptake is essential for T-ALL proliferation. Isotope tracing experiments showed that glutamine fuels aspartate synthesis through the TCA cycle and that glutamine and glutamine-derived aspartate together supply three nitrogen atoms in purines and all but one atom in pyrimidine rings. We show that the glutamate-aspartate transporter EAAT1 (SLC1A3), which is normally expressed in the central nervous system, is crucial for glutamine conversion to aspartate and nucleotides and that T-ALL cell proliferation depends on EAAT1 function. Through this work, we identify EAAT1 as a novel therapeutic target for T-ALL treatment.

Metabolomics in Cell Biology.

Metabolomics has long been used in a biomedical context. The most typical samples are body fluids in which small molecules can be detected and quantified using technologies such as Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). Many studies, in particular in the wider field of cancer research, are based on cellular models. Different cancer cells can have vastly different ways of regulating metabolism and responses to drug treatments depend on specific metabolic mechanisms which are often cell type specific. This has led to a series of publications using metabolomics to study metabolic mechanisms. Cell-based metabolomics has specific requirements and allows for interesting approaches where metabolism is followed in real-time. Here applications of metabolomics in cell biology have been reviewed, providing insight into specific technologies used and showing exemplary case studies with an emphasis towards applications which help to understand drug mechanisms.

Serum Metabolomic Profiling of Patients with Lipedema.

Lipedema is a chronic condition characterized by disproportionate and symmetrical enlargement of adipose tissue, predominantly affecting the lower limbs of women. This study investigated the use of metabolomics in lipedema research, with the objective of identifying complex metabolic disturbances and potential biomarkers for early detection, prognosis, and treatment strategies. The study group (n = 25) comprised women diagnosed with lipedema. The controls were 25 lean women and 25 obese females, both matched for age. In the patients with lipedema, there were notable changes in the metabolite parameters. Specifically, lower levels of histidine and phenylalanine were observed, whereas pyruvic acid was elevated compared with the weight controls. The receiver operating characteristic (ROC) curves for the diagnostic accuracy of histidine, phenylalanine, and pyruvic acid concentrations in distinguishing between patients with lipedema and those with obesity but without lipedema revealed good diagnostic ability for all parameters, with pyruvic acid being the most promising (area under the curve (AUC): 0.9992). Subgroup analysis within matched body mass index (BMI) ranges (30.0 to 39.9 kg/m2) further revealed that differences in pyruvic acid, phenylalanine, and histidine levels are likely linked to lipedema pathology rather than BMI variations. Changes in low-density lipoprotein (LDL)-6 TG levels and significant reductions in various LDL-2-carried lipids of patients with lipedema, compared with the lean controls, were observed. However, these lipids were similar between the lipedema patients and the obese controls, suggesting that these alterations are related to adiposity. Metabolomics is a valuable tool for investigating lipedema, offering a comprehensive view of metabolic changes and insights into lipedema's underlying mechanisms.

Towards a Precise NMR Quantification of Acute Phase Inflammation Proteins from Human Serum.

Nuclear Magnetic Resonance (NMR) spectra of human serum and plasma show, besides metabolites and lipoproteins, two characteristic signals termed GlycA and B arising from the acetyl groups of glycoprotein glycans from acute phase proteins, which constitute good markers for inflammatory processes. Here, we report a comprehensive assignment of glycoprotein glycan NMR signals observed in human serum, showing that GlycA and GlycB signals originate from Neu5Ac and GlcNAc moieties from N-glycans, respectively. Diffusion-edited NMR experiments demonstrate that signal components can be associated with specific acute phase proteins. Conventionally determined concentrations of acute phase glycoproteins correlate well with distinct features in NMR spectra (R2 up to 0.9422, p-value <0.001), allowing the simultaneous quantification of several acute phase inflammation proteins. Overall, a proteo-metabolomics NMR signature of significant diagnostic potential is obtained within 10-20 min acquisition time. This is exemplified in serum samples from COVID-19 and cardiogenic shock patients showing significant changes in several acute phase proteins compared to healthy controls.

TKTL1 Knockdown Impairs Hypoxia-Induced Glucose-6-phosphate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase Overexpression.

Increased expression of transketolase (TKT) and its isoform transketolase-like-1 (TKTL1) has been related to the malignant leukemia phenotype through promoting an increase in the non-oxidative branch of the pentose phosphate pathway (PPP). Recently, it has also been described that TKTL1 can have a role in survival under hypoxic conditions and in the acquisition of radio resistance. However, TKTL1's role in triggering metabolic reprogramming under hypoxia in leukemia cells has never been characterized. Using THP-1 AML cells, and by combining metabolomics and transcriptomics techniques, we characterized the impact of TKTL1 knockdown on the metabolic reprogramming triggered by hypoxia. Results demonstrated that TKTL1 knockdown results in a decrease in TKT, glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities and impairs the hypoxia-induced overexpression of G6PD and GAPDH, all having significant impacts on the redox capacity of NADPH- and NADH-related cells. Moreover, TKTL1 knockdown impedes hypoxia-induced transcription of genes encoding key enzymes and transporters involved in glucose, PPP and amino acid metabolism, rendering cells unable to switch to enhanced glycolysis under hypoxia. Altogether, our results show that TKTL1 plays a key role in the metabolic adaptation to hypoxia in THP-1 AML cells through modulation of G6PD and GAPDH activities, both regulating glucose/glutamine consumption and the transcriptomic overexpression of key players of PPP, glucose and amino acids metabolism.

Real-Time NMR Spectroscopy for Studying Metabolism.

Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in-cell NMR spectroscopy experiments. We are able to monitor real-time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer-based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.

Quantitative Isotopomer Rates in Real-Time Metabolism of Cells Determined by NMR Methods.

Tracer-based metabolism is becoming increasingly important for studying metabolic mechanisms in cells. NMR spectroscopy offers several approaches to measure label incorporation in metabolites, including 13 C- and 1 H-detected spectra. The latter are generally more sensitive, but quantification depends on the proton-carbon 1 JCH coupling constant, which varies significantly between different metabolites. It is therefore not possible to have one experiment optimised for all metabolites, and quantification of 1 H-edited spectra such as HSQCs requires precise knowledge of coupling constants. Increasing interest in tracer-based and metabolic flux analysis requires robust analyses with reasonably small acquisition times. Herein, we compare 13 C-filtered and 13 C-edited methods for quantification and show the applicability of the methods for real-time NMR spectroscopy of cancer-cell metabolism, in which label incorporations are subject to constant flux. We find an approach using a double filter to be most suitable and sufficiently robust to reliably obtain 13 C incorporations from difference spectra. This is demonstrated for JJN3 multiple myeloma cells processing glucose over 24 h. The proposed method is equally well suited for calculating the level of label incorporation in labelled cell extracts in the context of metabolic flux analysis.

nmrML: A Community Supported Open Data Standard for the Description, Storage, and Exchange of NMR Data.

NMR is a widely used analytical technique with a growing number of repositories available. As a result, demands for a vendor-agnostic, open data format for long-term archiving of NMR data have emerged with the aim to ease and encourage sharing, comparison, and reuse of NMR data. Here we present nmrML, an open XML-based exchange and storage format for NMR spectral data. The nmrML format is intended to be fully compatible with existing NMR data for chemical, biochemical, and metabolomics experiments. nmrML can capture raw NMR data, spectral data acquisition parameters, and where available spectral metadata, such as chemical structures associated with spectral assignments. The nmrML format is compatible with pure-compound NMR data for reference spectral libraries as well as NMR data from complex biomixtures, i.e., metabolomics experiments. To facilitate format conversions, we provide nmrML converters for Bruker, JEOL and Agilent/Varian vendor formats. In addition, easy-to-use Web-based spectral viewing, processing, and spectral assignment tools that read and write nmrML have been developed. Software libraries and Web services for data validation are available for tool developers and end-users. The nmrML format has already been adopted for capturing and disseminating NMR data for small molecules by several open source data processing tools and metabolomics reference spectral libraries, e.g., serving as storage format for the MetaboLights data repository. The nmrML open access data standard has been endorsed by the Metabolomics Standards Initiative (MSI), and we here encourage user participation and feedback to increase usability and make it a successful standard.

Combined Analysis of NMR and MS Spectra (CANMS).

Cellular metabolism in mammalian cells represents a challenge for analytical chemistry in the context of current biomedical research. Mass spectrometry and NMR spectroscopy together with computational tools have been used to study metabolism in cells. Compartmentalization of metabolism complicates the interpretation of stable isotope patterns in mammalian cells owing to the superimposition of different pathways contributing to the same pool of analytes. This indicates a need for a model-free approach to interpret such data. Mass spectrometry and NMR spectroscopy provide complementary analytical information on metabolites. Herein an approach that simulates 13 C multiplets in NMR spectra and utilizes mass increments to obtain long-range information is presented. The combined information is then utilized to derive isotopomer distributions. This is a first rigorous analytical and computational approach for a model-free analysis of metabolic data applicable to mammalian cells.

Metabolomics Biomarkers for Breast Cancer.

Metabolomics represents a more recent addition to the range of omics tools, which are increasingly used in clinical applications. By measuring the composition of small molecules in tissues, blood or urine, it provides a sensitive molecular readout often associated with disease and its states, especially in cancer. Changes in metabolism related to cancer are increasingly well understood and are seen as a major hallmark of cancer. This review covers metabolomics used in human breast cancers, with a focus on its application in clinical diagnostics. There are clear indications that metabolomics could be a useful addition to currently established clinical diagnostic tools for breast cancer, including the possibility to detect cancer and to predict treatment responses and survival rates from blood and tissue samples.

Metabolomic Analysis of Diapausing and Noni-diapausing Larvae of the European Corn Borer Ostrinia nubilalis (Hbn.) (Lepidoptera: Crambidae).

In this study, an (1)H-NMR -based metabolomic approach was used to investigate the biochemical mechanisms of diapause and cold hardiness in diapausing larvae of the European corn borer Ostrinia nubilalis. Metabolomic patterns in polar hemolymph extracts from non-diapausing and diapausing larvae of O. nubilalis were compared. Analysis indicated 13 metabolites: 7 amino acids, glycerol, acetate, citrate, succinate, lactate and putrescine. Results show that diapausing larvae display different metabolomic patterns compared to active non-diapausing larvae, with predominant metabolites identified as glycerol, proline and alanine. In specific diapausing larvae initially kept at 5 °C then gradually chilled to ­3 °C and ­16 °C, alanine , glycerol and acetate were predominant metabolites. (1)H-NMR spectroscopy provides new insight into the metabolomic patterns associated with cold resistance and diapause in O. nubilalis larvae, suggesting distinct metabolomes function in actively developing and diapausing larvae.

Distinct metabolomic and lipidomic profiles in serum samples of patients with primary sclerosing cholangitis.

Intoduction: Identification of specific metabolome and lipidome profile of patients with primary sclerosing cholangitis (PSC) is crucial for diagnosis, targeted personalized therapy, and more accurate risk stratification. Methods: Nuclear magnetic resonance (NMR) spectroscopy revealed an altered metabolome and lipidome of 33 patients with PSC [24 patients with inflammatory bowel disease (IBD) and 9 patients without IBD] compared with 40 age-, sex-, and body mass index (BMI)-matched healthy controls (HC) as well as 64 patients with IBD and other extraintestinal manifestations (EIM) but without PSC. Results: In particular, higher concentrations of pyruvic acid and several lipoprotein subfractions were measured in PSC in comparison to HC. Of clinical relevance, a specific amino acid and lipid profile was determined in PSC compared with IBD and other EIM. Discussion: These results have the potential to improve diagnosis by differentiating PSC patients from HC and those with IBD and EIM.

Dynamic nuclear hyperpolarization in liquids.

Nuclear magnetic resonance (NMR) spectroscopy is a broadly used analytical method with major applications in chemistry, biochemistry and medicine. Key applications include structural analysis of small molecules, metabolites, larger biomolecules such as proteins, RNA and DNA, and applications in material science. Magnetic resonance imaging (MRI), which is based on the same physical principles, is extensively used in medical diagnostics and represents the most widespread application of NMR. However, NMR is fundamentally limited in sensitivity and this has always restricted its applicability. Hyperpolarization techniques such as dynamic nuclear polarization (DNP) have become a major field of research and development because they hold the promise of increasing the sensitivity of NMR by several orders of magnitude. Such sensitivity enhancements could significantly broaden NMR applications, combining its unique structural information with much higher sensitivity. Unfortunately, there is no single implementation of DNP that would be suitable for a broader range of typical NMR applications. Experimental conditions often circumscribe areas of possible applications. Nevertheless, recent developments point towards experimental protocols providing solutions for specific applications of NMR. This review summarizes the concepts behind DNP in the light of recent developments and potential applications.

Quantum rotor induced hyperpolarization.

Despite its broad applicability NMR has always been limited by its inherently low sensitivity. Hyperpolarization methods have the potential to overcome this limitation and, in the case of ex situ dynamic nuclear polarization (DNP), large enhancement factors have been achieved. Although many other polarization methods have been described in the past, including chemically and parahydrogen-induced polarization and optical pumping, DNP has recently been the most popular. Here we present an additional polarization mechanism arising from quantum rotor effects in methyl groups, which generates polarizations at temperatures < 1.5 K and interferes with DNP at such temperatures. The polarization generated by this mechanism is efficiently transferred via carbon bound protons. Although quantum rotor polarizations have been studied for a small range of molecules in great detail, we observe such effects for a much broader range of substances with very different polarization rates at temperatures < 1.5 K. Moreover, we report transfer of quantum rotor polarization across a chain of protons. The observed effect not only influences the polarization in low-temperature DNP experiments but also opens a new independent avenue to generate enhanced sensitivity for NMR.

Tracer-Based Metabolic Analysis by NMR in Intact Perfused Human Liver Tissue.

Human metabolic liver disease is dramatically increasing globally and presents an urgent clinical unmet need. Rodent models of non-alcoholic fatty liver disease (NAFLD) are available, but they fail to fully recreate the metabolic and cellular features of human disease. Thus, it is imperative to understand the metabolic interplay in human cells in the context of disease. We have applied nuclear magnetic resonance (NMR) spectroscopy approaches to enable the detection of numerous metabolites in human cells and within intact tissue in a single measurement. In this chapter, we describe the challenges of using isolated human hepatocytes vs perfused human liver tissue for metabolic tracer experiments and how experimental parameters can be refined to interrogate signals from intact tissue and cells.

Food Fingerprinting: LC-ESI-IM-QTOF-Based Identification of Blumeatin as a New Marker Metabolite for the Detection of Origanum majorana Admixtures to O. onites/vulgare.

Oregano (Origanum vulgare and O. onites) is one of the most frequently counterfeited herbs in the world and is diluted with the leaves of a wide variety of plants. In addition to olive leaves, marjoram (O. majorana) is often used for this purpose in order to achieve a higher profit. However, apart from arbutin, no marker metabolites are known to reliably detect marjoram admixtures in oregano batches at low concentrations. In addition, arbutin is relatively widespread in the plant kingdom, which is why it is of great relevance to look for further marker metabolites in order to secure the analysis accordingly. Therefore, the aim of the present study was to use a metabolomics-based approach to identify additional marker metabolites with the aid of an ion mobility mass spectrometry instrument. The focus of the analysis was on the detection of non-polar metabolites, as this study was preceded by nuclear magnetic resonance spectroscopic investigations of the same samples based mainly on the detection of polar analytes. Using the MS-based approach, numerous marjoram specific features could be detected in admixtures of marjoram >10% in oregano. However, only one feature was detectable in admixtures of >5% marjoram. This feature was identified as blumeatin, which belongs to the class of flavonoid compounds. Initially, blumeatin was identified based on MS/MS spectra and collision cross section values using a database search. In addition, the identification of blumeatin was confirmed by a reference standard. Moreover, dried leaves of olive, myrtle, thyme, sage and peppermint, which are also known to be used to adulterate oregano, were measured. Blumeatin could not be detected in these plants, so this substance can be considered as an excellent marker compound for the detection of marjoram admixtures.

Unique Metabolomic and Lipidomic Profile in Serum From Patients With Crohn's Disease and Ulcerative Colitis Compared With Healthy Control Individuals.

BACKGROUND: Accurate biomarkers for disease activity and progression in patients with inflammatory bowel disease (IBD) are a prerequisite for individual disease characterization and personalized therapy. We show that metabolic profiling of serum from IBD patients is a promising approach to establish biomarkers. The aim of this work was to characterize metabolomic and lipidomic serum profiles of IBD patients in order to identify metabolic fingerprints unique to the disease. METHODS: Serum samples were obtained from 55 patients with Crohn's disease (CD), 34 patients with ulcerative colitis (UC), and 40 healthy control (HC) individuals and analyzed using proton nuclear magnetic resonance spectroscopy. Classification of patients and HC individuals was achieved by orthogonal partial least squares discriminant analysis and univariate analysis approaches. Disease activity was assessed using the Gastrointestinal Symptom Rating Scale. RESULTS: Serum metabolome significantly differed between CD patients, UC patients, and HC individuals. The metabolomic differences of UC and CD patients compared with HC individuals were more pronounced than the differences between UC and CD patients. Differences in serum levels of pyruvic acid, histidine, and the branched-chain amino acids leucine and valine were detected. The size of low-density lipoprotein particles shifted from large to small dense particles in patients with CD. Of note, apolipoprotein A1 and A2 serum levels were decreased in CD and UC patients with higher fecal calprotectin levels. The Gastrointestinal Symptom Rating Scale is negatively associated with the concentration of apolipoprotein A2. CONCLUSIONS: Metabolomic assessment of serum samples facilitated the differentiation of IBD patients and HC individuals. These differences were constituted by changes in amino acid and lipoprotein levels. Furthermore, disease activity in IBD patients was associated with decreased levels of the atheroprotective apolipoproteins A1 and A2.

The metabolic and lipidomic serum profile of patients with inflammatory bowel disease was analyzed using proton nuclear magnetic resonance spectroscopy. A significantly altered profile in comparison with healthy control individuals was identified, characterized by more atherogenic properties.

Modulation of structure and dynamics by disulfide bond formation in unfolded states.

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.

Crosstalk between AML and stromal cells triggers acetate secretion through the metabolic rewiring of stromal cells.

Acute myeloid leukaemia (AML) cells interact and modulate components of their surrounding microenvironment into their own benefit. Stromal cells have been shown to support AML survival and progression through various mechanisms. Nonetheless, whether AML cells could establish beneficial metabolic interactions with stromal cells is underexplored. By using a combination of human AML cell lines and AML patient samples together with mouse stromal cells and a MLL-AF9 mouse model, here we identify a novel metabolic crosstalk between AML and stromal cells where AML cells prompt stromal cells to secrete acetate for their own consumption to feed the tricarboxylic acid cycle (TCA) and lipid biosynthesis. By performing transcriptome analysis and tracer-based metabolic NMR analysis, we observe that stromal cells present a higher rate of glycolysis when co-cultured with AML cells. We also find that acetate in stromal cells is derived from pyruvate via chemical conversion under the influence of reactive oxygen species (ROS) following ROS transfer from AML to stromal cells via gap junctions. Overall, we present a unique metabolic communication between AML and stromal cells and propose two different molecular targets, ACSS2 and gap junctions, that could potentially be exploited for adjuvant therapy.