Alessia Lodi

Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy

Blood serum is commonly used for clinical diagnostics because its protein composition bears a wealth of information about the health of an organism. More recently the analysis of the small molecule composition, the metabolome, has received increased attention because the metabolite composition is influenced by many diseases, by the administration of drugs and toxins, and by the diet and life style of an individual. When nuclear magnetic resonance spectroscopy is used as an analytical tool it is often preferable to remove catalytically active proteins, in particular for longer measurements, because metabolite concentrations are otherwise in constant flux. Here we have compared different protocols for the separation of proteins and metabolites, including precipitation methods and ultrafiltration. Whereas most extraction methods involving protein precipitation deplete some metabolites, ultrafiltration is superior in retaining metabolite concentrations and offers excellent reproducibility. We also describe a new method to recover the hydrophobic fraction for ultrafiltration with good reproducibility.

Metabolomic profiling of drug responses in acute myeloid leukaemia cell lines.

Combined bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) exert unexpected antileukaemic activities against acute myeloid leukaemia (AML) and these activities are associated with the generation of reactive oxygen species (ROS) within the tumor cells. Although the generation of ROS by these drugs is supported by preceding studies including our own, the interrelationship between the cellular effects of the drugs and ROS generation is not well understood. Here we report the use of NMR metabolomic profiling to further study the effect of BEZ and MPA on three AML cell lines and to shed light on the underlying mechanism of action. For this we focused on drug effects induced during the initial 24 hours of treatment prior to the onset of overt cellular responses and examined these in the context of basal differences in metabolic profiles between the cell lines. Despite their ultimately profound cellular effects, the early changes in metabolic profiles engendered by these drugs were less pronounced than the constitutive metabolic differences between cell types. Nonetheless, drug treatments engendered common metabolic changes, most markedly in the response to the combination of BEZ and MPA. These responses included changes to TCA cycle intermediates consistent with recently identified chemical actions of ROS. Notable amongst these was the conversion of α-ketoglutarate to succinate which was recapitulated by the treatment of cell extracts with exogenous hydrogen peroxide. These findings indicate that the actions of combined BEZ and MPA against AML cells are indeed mediated downstream of the generation of ROS rather than some hitherto unsuspected mechanism. Moreover, our findings demonstrate that metabolite profiles represent highly sensitive markers for genomic differences between cells and their responses to external stimuli. This opens new perspectives to use metabolic profiling as a tool to study the rational redeployment of drugs in new disease settings.

Combined Bezafibrate and Medroxyprogesterone Acetate: Potential Novel Therapy for Acute Myeloid Leukaemia

Background The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis. Principal Findings Here we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D2 (PGD2) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Δ12,14 PGJ2 (15d-PGJ2). BEZ increased PGD2 synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ2 by inhibiting the PGD2 11β -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD2 to 9α11β-PGF2α. B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ2. Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors. Significance Collectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ2. These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML.

Critical Role of Glucose Metabolism in Rheumatoid Arthritis Fibroblast-like Synoviocytes

Up‐regulation of glucose metabolism has been implicated not only in tumor cell growth but also in immune cells upon activation. However, little is known about the metabolite profile in rheumatoid arthritis (RA), particularly in fibroblast‐like synoviocytes (FLS). This study was undertaken to evaluate whether changes in glucose metabolism in RA FLS could play a role in inflammation and joint damage.

Effects of the application of different window functions and projection methods on processing of 1H J-resolved nuclear magnetic resonance spectra for metabolomics

Two dimensional (2D) homonuclear (1)H J-resolved (JRES) nuclear magnetic resonance spectroscopy is increasingly used in metabolomics. This approach visualises metabolite chemical shifts and scalar couplings along different spectral dimensions, thereby increasing peak dispersion and facilitating spectral assignments and accurate quantification. Here, we optimise the processing of 2D JRES spectra by evaluating different window functions, a traditional sine-bell (SINE) and a combined sine-bell-exponential (SEM) function. Furthermore, we evaluate different projection methods for generating 1D projected spectra (pJRES). Spectra were recorded from three disparate types of biological samples and evaluated in terms of sensitivity, reproducibility and resolution. Overall, the SEM window function yielded considerably higher sensitivity and comparable spectral reproducibility and resolution compared to SINE, for both 1D pJRES and 2D JRES datasets. Furthermore, for pJRES spectra, the highest spectral quality was obtained using SEM combined with skyline projection. These improvements lend further support to utilising 2D J-resolved spectroscopy in metabolomics.

Critical role of fibroblast‐like synoviocytes glycolytic metabolism in rheumatoid arthritis

Up‐regulation of glucose metabolism has been implicated not only in tumor cell growth but also in immune cells upon activation. However, little is known about the metabolite profile in rheumatoid arthritis (RA), particularly in fibroblast‐like synoviocytes (FLS). This study was undertaken to evaluate whether changes in glucose metabolism in RA FLS could play a role in inflammation and joint damage.

Proton NMR-based metabolite analyses of archived serial paired serum and urine samples from myeloma patients at different stages of disease activity identifies acetylcarnitine as a novel marker of active disease.

Background Biomarker identification is becoming increasingly important for the development of personalized or stratified therapies. Metabolomics yields biomarkers indicative of phenotype that can be used to characterize transitions between health and disease, disease progression and therapeutic responses. The desire to reproducibly detect ever greater numbers of metabolites at ever diminishing levels has naturally nurtured advances in best practice for sample procurement, storage and analysis. Reciprocally, since many of the available extensive clinical archives were established prior to the metabolomics era and were not processed in such an ‘ideal’ fashion, considerable scepticism has arisen as to their value for metabolomic analysis. Here we have challenged that paradigm. Methods We performed proton nuclear magnetic resonance spectroscopy-based metabolomics on blood serum and urine samples from 32 patients representative of a total cohort of 1970 multiple myeloma patients entered into the United Kingdom Medical Research Council Myeloma IX trial. Findings Using serial paired blood and urine samples we detected metabolite profiles that associated with diagnosis, post-treatment remission and disease progression. These studies identified carnitine and acetylcarnitine as novel potential biomarkers of active disease both at diagnosis and relapse and as a mediator of disease associated pathologies. Conclusions These findings show that samples conventionally processed and archived can provide useful metabolomic information that has important implications for understanding the biology of myeloma, discovering new therapies and identifying biomarkers potentially useful in deciding the choice and application of therapy.

Choline kinase inhibition in rheumatoid arthritis

Objectives Little is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer invasiveness. Aggressive behaviour of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) led us to evaluate whether this metabolic pathway could play a role in RA FLS function and joint damage. Methods Choline metabolic profile of FLS cells was determined by 1H magnetic resonance spectroscopy (1HMRS) under conditions of ChoKα inhibition. FLS function was evaluated using the ChoKα inhibitor MN58b (IC50=4.2 μM). For arthritis experiments, mice were injected with K/BxN sera. MN58b (3 mg/kg) was injected daily intraperitoneal beginning on day 0 or day 4 after serum administration. Results The enzyme is expressed in synovial tissue and in cultured RA FLS. Tumour necrosis factor (TNF) and platelet-derived growth factor (PDGF) stimulation increased ChoKα expression and levels of phosphocholine in FLS measured by Western Blot (WB) and metabolomic studies of choline-containing compounds in cultured RA FLS extracts respectively, suggesting activation of this pathway in RA synovial environment. A ChoKα inhibitor also suppressed the behaviour of cultured FLS, including cell migration and resistance to apoptosis, which might contribute to cartilage destruction in RA. In a passive K/BxN arthritis model, pharmacologic ChoKα inhibition significantly decreased arthritis in pretreatment protocols as well as in established disease. Conclusions These data suggest that ChoKα inhibition could be an effective strategy in inflammatory arthritis. It also suggests that targeting the metabolome can be a new treatment strategy in non-cancer conditions.

Hypoxia Triggers Major Metabolic Changes in AML Cells without Altering Indomethacin-Induced TCA Cycle Deregulation

Our previous studies have shown that the nonsteroidal anti-inflammatory drug indomethacin exhibits antileukemic activity in vitro and can inhibit the aldo-keto reductase AKR1C3, which we identified as a novel target in acute myeloid leukemia. However, the antileukemic actions of indomethacin are likely to be complex and extend beyond inhibition of either AKR1C3 or cycloxygenases. To further understand the antileukemic activity of indomethacin we have used untargeted nuclear magnetic resonance-based metabolic analysis to characterize the responses of KG1a and K562 cell lines in both normal culture conditions and in hypoxia, which better represents the tumor environment in vivo. Hypoxia induced dramatic metabolic changes in untreated KG1a and K562, including adaptation of both phospholipid and glycolytic metabolism. Despite these changes, both cell lines sustained relatively unaltered mitochondrial respiration. The administration of indomethacin induced similar metabolic responses regardless of the oxygen level in the environment. Notable exceptions included metabolites associated with de novo fatty acid synthesis and choline phospholipid metabolism. Collectively, these results suggest that leukemia cells have the inherent ability to tolerate changes in oxygen tension while maintaining an unaltered mitochondrial respiration. However, the administration of indomethacin significantly increased oxidative stress in both KG1a and K562, inducing mitochondrial dysfunction, regardless of the oxygenation conditions. These findings emphasize the particular pertinence of the tricarboxylic acid cycle to the survival of cancer cells and may explain why some antileukemic drugs have been discovered and developed successfully despite the use of culture conditions that do not reflect the hypoxic environment of cancer cells in vivo.

Inhibiting glutaminase in acute myeloid leukemia: metabolic dependency of selected AML subtypes

Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.