Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.

Increased cysteine metabolism in PINK1 models of Parkinson's disease.

Parkinson's disease (PD), an age-dependent neurodegenerative disease, is characterised by the selective loss of dopaminergic neurons in the substantia nigra (SN). Mitochondrial dysfunction is a hallmark of PD, and mutations in PINK1, a gene necessary for mitochondrial fitness, cause PD. Drosophila melanogaster flies with pink1 mutations exhibit mitochondrial defects and dopaminergic cell loss and are used as a PD model. To gain an integrated view of the cellular changes caused by defects in the PINK1 pathway of mitochondrial quality control, we combined metabolomics and transcriptomics analysis in pink1-mutant flies with human induced pluripotent stem cell (iPSC)-derived neural precursor cells (NPCs) with a PINK1 mutation. We observed alterations in cysteine metabolism in both the fly and human PD models. Mitochondrial dysfunction in the NPCs resulted in changes in several metabolites that are linked to cysteine synthesis and increased glutathione levels. We conclude that alterations in cysteine metabolism may compensate for increased oxidative stress in PD, revealing a unifying mechanism of early-stage PD pathology that may be targeted for drug development. This article has an associated First Person interview with the first author of the paper.

Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance.

PURPOSE: PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. EXPERIMENTAL DESIGN: We analyzed breast and ovarian patient-derived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. RESULTS: Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. CONCLUSIONS: Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi.

Hepatic Metabolic Profiling of Lifelong Exercise Training Rats.

Regular physical exercise has been investigated as a primary preventive measure of several chronic diseases and premature death. Moreover, it has been shown to synchronize responses across multiple organs. In particular, hepatic tissue has proven to be a descriptive matrix to monitor the effect of physical activity. In this study, we performed an untargeted metabolomics-based analysis of hepatic tissue extracts from rats that have undergone either lifelong or chronic exercise training. For this purpose, 56 hepatic samples were collected and were analyzed by UHPLC-TOF-MS in negative ionization mode. This approach involved untargeted metabolite detection on hepatic tissue extracts accompanied by an in-house retention time/accurate mass library enabling confident metabolite identification. Unsupervised (PCA) and supervised (OPLS-DA) multivariate analysis showed significant metabolic perturbation on a panel of 28 metabolites, including amino acids, vitamins, nucleotides, and sugars. The training regime employed in this study resulted in a probable acceleration of the bioenergetic processes (glycolysis, glycogen metabolism), promoted catabolism of purines, and supplied biosynthetic precursors via the pentose phosphate pathway and pentose and glucuronate interconversions. Overall, the applied methodology was able to discriminate the different training schedules based on the rat liver metabolome.

Metabolomic Profile in HFpEF vs HFrEF Patients.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) and HF with reduced ejection fraction (HFrEF) are associated with metabolic derangements, which may have different pathophysiological implications. METHODS AND RESULTS: In new-onset HFpEF (EF of ≥50%, n = 46) and HFrEF (EF of <40%, n = 75) patients, 109 endogenous plasma metabolites including amino acids, phospholipids and acylcarnitines were assessed using targeted metabolomics. Differentially altered metabolites and associations with clinical characteristics were explored. Patients with HFpEF were older, more often female with hypertension, atrial fibrillation, and diabetes compared with patients with HFrEF. Patients with HFpEF displayed higher levels of hydroxyproline and symmetric dimethyl arginine, alanine, cystine, and kynurenine reflecting fibrosis, inflammation and oxidative stress. Serine, cGMP, cAMP, l-carnitine, lysophophatidylcholine (18:2), lactate, and arginine were lower compared with patients with HFrEF. In patients with HFpEF with diabetes, kynurenine was higher (P = .014) and arginine lower (P = .014) vs patients with no diabetes, but did not differ with diabetes status in HFrEF. Decreasing kynurenine was associated with higher eGFR only in HFpEF (Pinteraction = .020). CONCLUSIONS: Patients with new-onset HFpEF compared with patients with new-onset HFrEF display a different metabolic profile associated with comorbidities, such as diabetes and kidney dysfunction. HFpEF is associated with indices of increased inflammation and oxidative stress, impaired lipid metabolism, increased collagen synthesis, and downregulated nitric oxide signaling. Together, these findings suggest a more predominant systemic microvascular endothelial dysfunction and inflammation linked to increased fibrosis in HFpEF compared with HFrEF. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03671122 https://clinicaltrials.gov.

Ion Pair Chromatography for Endogenous Metabolites LC-MS Analysis in Tissue Samples Following Targeted Acquisition.

A protocol for the preparation of tissue extracts for the targeted analysis of ca. 150 polar metabolites, including those involved in central carbon metabolism is described, using a reversed-phase ion pair U(H)PLC-MS method. Data collection enabled by multiple-reaction monitoring provides highly specific, sensitive acquisition of metabolic intermediates with a wide range of physicochemical properties and pathway coverage. Technical aspects are discussed for method transfer along with the basic principles of sample sequence setup, data analysis, and validation. General comments are given to help the assessment of data quality and system performance.

Quantitation of Endogenous Metabolites in Mouse Tumors Using Mass-Spectrometry Imaging.

Described is a quantitative-mass-spectrometry-imaging (qMSI) methodology for the analysis of lactate and glutamate distributions in order to delineate heterogeneity among mouse tumor models used to support drug-discovery efficacy testing. We evaluate and report on preanalysis-stabilization methods aimed at improving the reproducibility and efficiency of quantitative assessments of endogenous molecules in tissues. Stability experiments demonstrate that optimum stabilization protocols consist of frozen-tissue embedding, post-tissue-sectioning desiccation, and storage at -80 °C of tissue sections sealed in vacuum-tight containers. Optimized stabilization protocols are used in combination with qMSI methodology for the absolute quantitation of lactate and glutamate in tumors, incorporating the use of two different stable-isotope-labeled versions of each analyte and spectral-clustering performed on each tissue section using k-means clustering to allow region-specific, pixel-by-pixel quantitation. Region-specific qMSI was used to screen different tumor models and identify a phenotype that has low lactate heterogeneity, which will enable accurate measurements of lactate modulation in future drug-discovery studies. We conclude that using optimized qMSI protocols, it is possible to quantify endogenous metabolites within tumors, and region-specific quantitation can provide valuable insight into tissue heterogeneity and the tumor microenvironment.

Inhibiting PI3Kß with AZD8186 Regulates Key Metabolic Pathways in PTEN-Null Tumors.

Purpose: PTEN-null tumors become dependent on the PI3Kß isoform and can be targeted by molecules such as the selective PI3Kß inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, the consequences of inhibiting PI3Kß are poorly defined.Experimental Design: To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models.Results: AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kß also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis.Conclusions: This study identifies novel mechanistic biomarkers of PI3Kß inhibition in PTEN-null tumors supporting the concept that targeting PI3Kß may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents. Clin Cancer Res; 23(24); 7584-95. ©2017 AACR.

Pre-clinical pharmacology of AZD3965, a selective inhibitor of MCT1: DLBCL, NHL and Burkitt's lymphoma anti-tumor activity.

Tumors frequently display a glycolytic phenotype with increased flux through glycolysis and concomitant synthesis of lactate. To maintain glycolytic flux and prevent intracellular acidification, tumors efflux lactate via lactate transporters (MCT1-4). Inhibitors of lactate transport have the potential to inhibit glycolysis and tumor growth. We developed a small molecule inhibitor of MCT1 (AZD3965) and assessed its activity across a panel of cell lines. We explored its antitumor activity as monotherapy and in combination with doxorubicin or rituximab. AZD3965 is a potent inhibitor of MCT1 with activity against MCT2 but selectivity over MCT3 and MCT4. In vitro, AZD3965 inhibited the growth of a range of cell lines especially haematological cells. Inhibition of MCT1 by AZD3965 inhibited lactate efflux and resulted in accumulation of glycolytic intermediates. In vivo, AZD3965 caused lactate accumulation in the Raji Burkitt's lymphoma model and significant tumor growth inhibition. Moreover, AZD3965 can be combined with doxorubicin or rituximab, components of the R-CHOP standard-of-care in DLBCL and Burkitt's lymphoma. Finally, combining lactate transport inhibition by AZD3965 with GLS1 inhibition in vitro, enhanced cell growth inhibition and cell death compared to monotherapy treatment. The ability to combine AZD3965 with novel, and standard-of-care inhibitors offers novel combination opportunities in haematological cancers.

Targeted Metabolic Profiling of the Tg197 Mouse Model Reveals Itaconic Acid as a Marker of Rheumatoid Arthritis.

Rheumatoid arthritis is a progressive, highly debilitating disease where early diagnosis, enabling rapid clinical intervention, would provide obvious benefits to patients, healthcare systems, and society. Novel biomarkers that enable noninvasive early diagnosis of the onset and progression of the disease provide one route to achieving this goal. Here a metabolic profiling method has been applied to investigate disease development in the Tg197 arthritis mouse model. Hind limb extract profiling demonstrated clear differences in metabolic phenotypes between control (wild type) and Tg197 transgenic mice and highlighted raised concentrations of itaconic acid as a potential marker of the disease. These changes in itaconic acid concentrations were moderated or indeed reversed when the Tg197 mice were treated with the anti-hTNF biologic infliximab (10 mg/kg twice weekly for 6 weeks). Further in vitro studies on synovial fibroblasts obtained from healthy wild-type, arthritic Tg197, and infliximab-treated Tg197 transgenic mice confirmed the association of itaconic acid with rheumatoid arthritis and disease-moderating drug effects. Preliminary indications of the potential value of itaconic acid as a translational biomarker were obtained when studies on K4IM human fibroblasts treated with hTNF showed an increase in the concentrations of this metabolite.

Leptin, BMI, and a Metabolic Gene Expression Signature Associated with Clinical Outcome to VEGF Inhibition in Colorectal Cancer.

VEGF (vascular endothelial growth factor) signaling inhibitors are widely used in different cancer types; however, patient selection remains a challenge. Analyses of samples from a phase III clinical trial in metastatic colorectal cancer testing chemotherapy versus chemotherapy with the small molecule VEGF receptors inhibitor cediranib identified circulating leptin levels, BMI, and a tumor metabolic and angiogenic gene expression signature associated with improved clinical outcome in patients treated with cediranib. Patients with a glycolytic and hypoxic/angiogenic profile were associated with increased benefit from cediranib, whereas patients with a high lipogenic, oxidative phosphorylation and serine biosynthesis signature did not gain benefit. These findings translated to pre-clinical tumor xenograft models where the same metabolic gene expression profiles were associated with in vivo sensitivity to cediranib as monotherapy. These findings suggest a link between patient physiology, tumor biology, and response to antiangiogenics, which may guide patient selection for VEGF therapy in the future.

Solid phase extraction methodology for UPLC-MS based metabolic profiling of urine samples.

In general, when performing untargeted metabolic phenotyping (metabolomics/metabonomics) studies on biological samples for example urine or food, sample preparation should be kept to a minimum. However, there are circumstances when desalting, preconcentration, or the fractionation of samples into polar and nonpolar metabolites is of value for enabling the subsequent analysis. Because of its simplicity and ease of automation SPE is well suited to such applications prior to analysis by ultra-performance LC-TOF-MS. In the present study, the properties of a range of SPE phases have been investigated with respect to the range of metabolites that can be extracted from urine. The phases include alkyl modified (C8 and, C18-OH and C18) silica and polymeric materials. The results show that the C18 phase was well suited to fractionating urine into samples suitable for separate analysis of polar and nonpolar constituents via HILIC and RPLC, respectively, while the polymeric materials were best for concentrating and desalting samples.

Inhibition of monocarboxylate transporter-1 (MCT1) by AZD3965 enhances radiosensitivity by reducing lactate transport.

Inhibition of the monocarboxylate transporter MCT1 by AZD3965 results in an increase in glycolysis in human tumor cell lines and xenografts. This is indicated by changes in the levels of specific glycolytic metabolites and in changes in glycolytic enzyme kinetics. These drug-induced metabolic changes translate into an inhibition of tumor growth in vivo. Thus, we combined AZD3965 with fractionated radiation to treat small cell lung cancer (SCLC) xenografts and showed that the combination provided a significantly greater therapeutic effect than the use of either modality alone. These results strongly support the notion of combining MCT1 inhibition with radiotherapy in the treatment of SCLC and other solid tumors.

Targeted profiling of polar intracellular metabolites using ion-pair-high performance liquid chromatography and -ultra high performance liquid chromatography coupled to tandem mass spectrometry: applications to serum, urine and tissue extracts.

The effective analysis of polar ionic metabolites by LC-MS, such as those encountered in central carbon metabolism, represents a major problem for metabolic profiling that is not adequately addressed using strategies based on either reversed-phase or HILIC methods. Here we have compared analysis of central carbon metabolites on optimized methods using HILIC, porous graphitic carbon or ion pair chromatography (IPC) using tributyl ammonium as IP reagent. Of the 3 chromatographic approaches examined only IPC enabled us to obtain a robust analytical methodology. This system was used to profile more than a hundred endogenous metabolic intermediates in urine, serum and tissue samples. However, whilst we found IPC to be the best of the approaches examined considerable care was still needed to obtain robust data. Thus, in excess of 40 of representative biological samples were needed to "condition" a new analytical column and further 10 matrix injections were then required at the beginning of each analytical batch in order to obtain robust and reproducible chromatographic separations. An additional limitation that we have found was that, for a small number of phosphorylated and poly carboxylic acid metabolites, measurement was only possible if the analytes were present in relatively high concentrations. We also found that, whilst this methodology could be used for the analysis of both in vitro cell culture media, cell extracts, tissue, and biological fluids (blood, urine), for the best results columns should only be used to analyze a single matrix. However, despite the need for extensive column conditioning, and the manifold disadvantages resulting from the contamination of the separation system and mass spectrometer with the ion pair reagent, IPC-MS currently provides the best means of analyzing these polar, ionic and problematic metabolites.

Endogenous and xenobiotic metabolite profiling of liver extracts from SCID and chimeric humanized mice following repeated oral administration of troglitazone.

1. Metabonomic analysis, via a combination of untargeted and targeted liquid chromatography-mass spectrometry (LC-MS) and untargeted (1)H NMR spectroscopy-based metabolite profiling, was performed on aqueous (AQ) and organic liver extracts from control (SCID) and chimeric humanized (PXB) mice dosed with troglitazone at 0, 300 and 600 mg/kg/day for seven days. 2. LC-MS analysis of AQ liver extracts showed a more "human-like" profile for troglitazone metabolites for PXB, compared with SCID, mice. 3. LC-MS detected differences in endogenous metabolites, particularly lipid species in dosed mice, including elevated triacylglycerols and 1-alkyl,2-acylglycerophosphates as well as lowered diacylglycerophosphocholines and 1-alkyl,2-acylglycerophosphocholines for PXB compared with SCID mouse liver extracts. Following drug administration changes in the relative proportions of the ions for various unsaturated fatty acids were observed for both types of mouse, some of which were specific to PXB or SCID mice. 4. (1)H NMR spectroscopy revealed that AQ PXB mouse liver extracts had elevated amounts of inosine, fumarate, creatine, aspartate, trimethylamine N-oxide, glycerophosphocholine, phosphocholine, choline, glutamine, glutamate, acetate, alanine and lactate relative to SCID mice and decreased histidine, glycogen, α- and ß-glucose, taurine, and glutathione. Increased uracil and tyrosine concentrations were detected for PXB mice on troglitazone administration. 5. Metabonomic profiling thus showed clear differences between humanized and SCID mice, including after administration of troglitazone.

Phosphorus and sulfur metabonomic profiling of tissue and plasma obtained from tumour-bearing mice using ultra-performance liquid chromatography/inductively coupled plasma mass spectrometry.

RATIONALE: Metabonomic studies use complex biological samples (blood plasma/serum, tissues, etc.) that when analysed with high-performance liquid chromatography/mass spectrometry (HPLC/MS) or nuclear magnetic resonance (NMR) generate profiles that may contain many thousands of features. These profiles can be difficult to interpret with the majority of the features contributing little to the study. As such there is an argument for the development of techniques that can simplify the problem by targeting particular classes of compounds. METHODS: In this study ultra-performance liquid chromatography/inductively coupled plasma mass spectrometry (UPLC/ICP-MS) was used to profile tumour tissue and plasma samples for phosphorus- and sulfur-containing metabolites. These samples were xenograft tumours (derived from breast, lung and colon cell lines) and plasma obtained from nude mice. Plasma was also obtained from non-tumour-bearing mice as a control. Due to isobaric interferences this method took advantage of the dynamic reaction cell within the ICP-MS system to react the phosphorus and sulfur ions with oxygen. The PO+ and SO+ ions were then monitored free of interferences. The total phosphorus and sulfur within each sample was also recorded using flow injection ICP-MS. A robust quality control system based on pooled sample replicate analysis was used throughout the study. RESULTS: Determination of the total phosphorus and sulfur content of each sample was sufficient in itself for statistical differentiation between the majority of the cell lines analysed. Subsequent reversed-phase chromatographic profiling of the organic tumour and plasma extracts revealed the presence of a number of well-retained phosphorus-containing compounds that showed tumour-specific profiles. Reversed-phase profiling was not suitable for the sulfur-containing compounds which eluted with the solvent front. CONCLUSIONS: This study has shown the potential use of UPLC/ICP-MS to differentiate between tumour cell lines, using both plasma and tumour tissue samples, based solely on metabolites that contain phosphorus or sulfur. Whilst further work is required to identify these compounds this methodology shows the ability of the described methods to provide targets for future biomarker discovery studies. Copyright © 2013 John Wiley & Sons, Ltd.

Global metabolic profiling of animal and human tissues via UPLC-MS.

Obtaining comprehensive, untargeted metabolic profiles for complex solid samples, e.g., animal tissues, requires sample preparation and access to information-rich analytical methodologies such as mass spectrometry (MS). Here we describe a practical two-step process for tissue samples that is based on extraction into 'aqueous' and 'organic' phases for polar and nonpolar metabolites. Separation methods such as ultraperformance liquid chromatography (UPLC) in combination with MS are needed to obtain sufficient resolution to create diagnostic metabolic profiles and identify candidate biomarkers. We provide detailed protocols for sample preparation, chromatographic procedures, multivariate analysis and metabolite identification via tandem MS (MS/MS) techniques and high-resolution MS. By using these optimized approaches, analysis of a set of samples using a 96-well plate format would take ~48 h: 1 h for system setup, 8-10 h for sample preparation, 34 h for UPLC-MS analysis and 2-3 h for preliminary/exploratory data processing, representing a robust method for untargeted metabolic screening of tissue samples.

Investigation of chronic alcohol consumption in rodents via ultra-high-performance liquid chromatography-mass spectrometry based metabolite profiling.

Alcohol consumption in man, when seen in its extreme form of alcoholism, is a complex and socially disruptive disorder that can result in significant levels of liver injury. Here the rodent "intragastric feeding model" was used together with UHPLC-TOFMS analysis to determine changes in global metabolite profiles for plasma and urine from alcohol treated rats and mice compared to control animals. Multivariate statistical analysis (using principal components analysis, PCA) revealed robust differences between profiles from control and alcohol-treated animals from both species. A large number of metabolites were seen to differ between control and alcohol-treated animals, for both biofluids.

Metabolite profiles from dried blood spots for metabonomic studies using UPLC combined with orthogonal acceleration ToF-MS: effects of different papers and sample storage stability.

BACKGROUND: Investigations have been undertaken of the effects of storage on the utility of using dried blood spots, collected and stored on a paper substrate, combined with reversed-phase ultra-performance LC (UPLC), electrospray ionization orthogonal acceleration ToF-MS (UPLC-oaToF-MS), as a method for performing global metabolic profiling studies. In addition, a range of products used to collect these blood spots have also been studied for effects on the UPLC-MS background. RESULTS: These preliminary data suggest that sample stability in this application is limited unless the cards are stored at, at least, -20°C and preferably at -80°C. In addition, the use of untreated cards is recommended to minimize incurred background interferences. CONCLUSION: Further investigations are necessary to improve the utility of this methodology in global metabolite profiling.