Srebp-controlled glucose metabolism is essential for NK cell functional responses.

Activated natural killer (NK) cells engage in a robust metabolic response that is required for normal effector function. Using genetic, pharmacological and metabolic analyses, we demonstrated an essential role for Srebp transcription factors in cytokine-induced metabolic reprogramming of NK cells that was independent of their conventional role in the control of lipid synthesis. Srebp was required for elevated glycolysis and oxidative phosphorylation and promoted a distinct metabolic pathway configuration in which glucose was metabolized to cytosolic citrate via the citrate-malate shuttle. Preventing the activation of Srebp or direct inhibition of the citrate-malate shuttle inhibited production of interferon-γ and NK cell cytotoxicity. Thus, Srebp controls glucose metabolism in NK cells, and this Srebp-dependent regulation is critical for NK cell effector function.

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs.

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.

Anomaly detection in mixed high-dimensional molecular data.

MOTIVATION: Mixed molecular data combines continuous and categorical features of the same samples, such as OMICS profiles with genotypes, diagnoses, or patient sex. Like all high-dimensional molecular data, it is prone to incorrect values that can stem from various sources for example the technical limitations of the measurement devices, errors in the sample preparation, or contamination. Most anomaly detection algorithms identify complete samples as outliers or anomalies. However, in most cases, not all measurements of those samples are erroneous but only a few one-dimensional features within the samples are incorrect. These one-dimensional data errors are continuous measurements that are either located outside or inside the normal ranges of their features but in both cases show atypical values given all other continuous and categorical features in the sample. Additionally, categorical anomalies can occur for example when the genotype or diagnosis was submitted wrongly. RESULTS: We introduce ADMIRE (Anomaly Detection using MIxed gRaphical modEls), a novel approach for the detection and correction of anomalies in mixed high-dimensional data. Hereby, we focus on the detection of single (one-dimensional) data errors in the categorical and continuous features of a sample. For that the joint distribution of continuous and categorical features is learned by mixed graphical models, anomalies are detected by the difference between measured and model-based estimations and are corrected using imputation. We evaluated ADMIRE in simulation and by screening for anomalies in one of our own metabolic datasets. In simulation experiments, ADMIRE outperformed the state-of-the-art methods of Local Outlier Factor, stray, and Isolation Forest. AVAILABILITY AND IMPLEMENTATION: All data and code is available at https://github.com/spang-lab/adadmire. ADMIRE is implemented in a Python package called adadmire which can be found at https://pypi.org/project/adadmire.

Associations of Urine and Plasma Metabolites with Kidney Failure and Death in a CKD Cohort.

RATIONALE & OBJECTIVE: Biomarkers that enable better identification of persons with chronic kidney disease (CKD) who are at higher risk for disease progression and adverse events are needed. This study sought to identify urine and plasma metabolites associated with progression of kidney disease. STUDY DESIGN: Prospective metabolome-wide association study. SETTING & PARTICIPANTS: Persons with CKD enrolled in the German CKD Study (GCKD) with metabolite measurements; with external validation within the Atherosclerosis Risk in Communities Study. EXPOSURES: 1,513 urine and 1,416 plasma metabolites (Metabolon, Inc.) measured at study entry using untargeted mass spectrometry. OUTCOMES: Main endpoints were kidney failure (KF), and a composite endpoint of KF, eGFR <15 mL/min/1.73m2, or 40% decline in eGFR (CKE). Death from any cause was a secondary endpoint. After a median of 6.5 years follow-up, 500 persons experienced KF, 1,083 experienced CKE and 680 died. ANALYTICAL APPROACH: Time-to-event analyses using multivariable proportional hazard regression models in a discovery-replication design, with external validation. RESULTS: 5,088 GCKD participants were included in analyses of urine metabolites and 5,144 in analyses of plasma metabolites. Among 182 unique metabolites, 30 were significantly associated with KF, 49 with CKE, and 163 with death. The strongest association with KF was observed for plasma hydroxyasparagine (hazard ratio: 1.95, 95% confidence interval: 1.68-2.25). An unnamed metabolite measured in plasma and urine was significantly associated with KF, CKE, and death. External validation of the identified associations of metabolites with KF or CKE revealed direction-consistency for 88% of observed associations. Selected associations of 18 metabolites with study outcomes have not been previously reported. LIMITATIONS: Use of observational data and semi-quantitative metabolite measurements at a single time point. CONCLUSIONS: The observed associations between metabolites and KF, CKE or death in persons with CKD confirmed previously reported findings and also revealed several associations not previously described. These findings warrant confirmatory research in other study cohorts.

D-2-hydroxyglutarate supports a tolerogenic phenotype with lowered major histocompatibility class II expression in non-malignant dendritic cells and acute myeloid leukemia cells.

D-2-hydroxyglutarate (D-2-HG) accumulates in primary acute myeloid leukemia (AML) patients with mutated isocitrate dehydrogenase (IDH) and other malignancies. D-2-HG suppresses antitumor T cell immunity but little is known about potential effects on non-malignant myeloid cells. Here we show that D-2-HG impairs human but not murine dendritic cell (DC) differentiation, resulting in a tolerogenic phenotype with low major histocompatibility (MHC) class II expression. In line, IDH-mutated AML blasts exhibited lower expression of HLA-DP and were less susceptible to lysis by HLA-DP-specific T cells. Interestingly, D-2-HG reprogrammed metabolism towards increased lactate production in DCs and AML besides its expected impact on DNA demethylation. Vitamin C accelerated DNA demethylation, but only the combination of vitamin C and glycolytic inhibition lowered lactate levels and supported MHC class II expression. Our results indicate an unexpected link between the immunosuppressive metabolites 2-HG and lactic acid and suggest a potentially novel therapeutic strategy with combinations of anti-glycolytic drugs and epigenetic modulators (hypomethylating agents) or other therapeutics for the treatment of AML.

A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells.

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo.

De novo polyamine synthesis supports metabolic and functional responses in activated murine NK cells.

Cellular metabolism is dynamically regulated in NK cells and strongly influences their responses. Metabolic dysfunction is linked to defective NK cell responses in diseases such as obesity and cancer. The transcription factors, sterol regulatory element binding protein (SREBP) and cMyc, are crucial for controlling NK cell metabolic and functional responses, though the mechanisms involved are not fully understood. This study reveals a new role for SREBP in NK cells in supporting de novo polyamine synthesis through facilitating elevated cMyc expression. Polyamines have diverse roles and their de novo synthesis is required for NK cell glycolytic and oxidative metabolism and to support optimal NK cell effector functions. When NK cells with impaired SREBP activity were supplemented with exogenous polyamines, NK cell metabolic defects were not rescued but these NK cells displayed significant improvement in some effector functions. One role for polyamines is in the control of protein translation where spermidine supports the posttranslational hypusination of translation factor eIF5a. Pharmacological inhibition of hypusination also impacts upon NK cell metabolism and effector function. Considering recent evidence that cholesterol-rich tumor microenvironments inhibit SREBP activation and drive lymphocyte dysfunction, this study provides key mechanistic insight into this tumor-evasion strategy.

Tachycardiomyopathy entails a dysfunctional pattern of interrelated mitochondrial functions.

Tachycardiomyopathy is characterised by reversible left ventricular dysfunction, provoked by rapid ventricular rate. While the knowledge of mitochondria advanced in most cardiomyopathies, mitochondrial functions await elucidation in tachycardiomyopathy. Pacemakers were implanted in 61 rabbits. Tachypacing was performed with 330 bpm for 10 days (n = 11, early left ventricular dysfunction) or with up to 380 bpm over 30 days (n = 24, tachycardiomyopathy, TCM). In n = 26, pacemakers remained inactive (SHAM). Left ventricular tissue was subjected to respirometry, metabolomics and acetylomics. Results were assessed for translational relevance using a human-based model: induced pluripotent stem cell derived cardiomyocytes underwent field stimulation for 7 days (TACH-iPSC-CM). TCM animals showed systolic dysfunction compared to SHAM (fractional shortening 37.8 ± 1.0% vs. 21.9 ± 1.2%, SHAM vs. TCM, p < 0.0001). Histology revealed cardiomyocyte hypertrophy (cross-sectional area 393.2 ± 14.5 µm2 vs. 538.9 ± 23.8 µm2, p < 0.001) without fibrosis. Mitochondria were shifted to the intercalated discs and enlarged. Mitochondrial membrane potential remained stable in TCM. The metabolite profiles of ELVD and TCM were characterised by profound depletion of tricarboxylic acid cycle intermediates. Redox balance was shifted towards a more oxidised state (ratio of reduced to oxidised nicotinamide adenine dinucleotide 10.5 ± 2.1 vs. 4.0 ± 0.8, p < 0.01). The mitochondrial acetylome remained largely unchanged. Neither TCM nor TACH-iPSC-CM showed relevantly increased levels of reactive oxygen species. Oxidative phosphorylation capacity of TCM decreased modestly in skinned fibres (168.9 ± 11.2 vs. 124.6 ± 11.45 pmol·O2·s-1·mg-1 tissue, p < 0.05), but it did not in isolated mitochondria. The pattern of mitochondrial dysfunctions detected in two models of tachycardiomyopathy diverges from previously published characteristic signs of other heart failure aetiologies.

A Predictive Model for Progression of CKD to Kidney Failure Based on Routine Laboratory Tests.

RATIONALE & OBJECTIVE: Stratification of chronic kidney disease (CKD) patients at risk for progressing to kidney failure requiring kidney replacement therapy (KFRT) is important for clinical decision-making and trial enrollment. STUDY DESIGN: Four independent prospective observational cohort studies. SETTING & PARTICIPANTS: The development cohort comprised 4,915 CKD patients, and 3 independent validation cohorts comprised a total of 3,063. Patients were observed for approximately 5 years. EXPOSURE: 22 demographic, anthropometric, and laboratory variables commonly assessed in CKD patients. OUTCOME: Progression to KFRT. ANALYTICAL APPROACH: A least absolute shrinkage and selection operator (LASSO) Cox proportional hazards model was fit to select laboratory variables that best identified patients at high risk for KFRT. Model discrimination and calibration were assessed and compared against the 4-variable Tangri (T4) risk equation both in a resampling approach within the development cohort and in the validation cohorts using cause-specific concordance (C) statistics, net reclassification improvement, and calibration graphs. RESULTS: The newly derived 6-variable risk score (Z6) included serum creatinine, albumin, cystatin C, and urea, as well as hemoglobin and the urinary albumin-creatinine ratio. In the the resampling approach, Z6 achieved a median C statistic of 0.909 (95% CI, 0.868-0.937) at 2 years after the baseline visit, whereas the T4 achieved a median C statistic of 0.855 (95% CI, 0.799-0.915). In the 3 independent validation cohorts, the Z6C statistics were 0.894, 0.921, and 0.891, whereas the T4C statistics were 0.882, 0.913, and 0.862. LIMITATIONS: The Z6 was both derived and tested only in White European cohorts. CONCLUSIONS: A new risk equation based on 6 routinely available laboratory tests facilitates identification of patients with CKD who are at high risk of progressing to KFRT.

A serum microRNA sequence reveals fragile X protein pathology in amyotrophic lateral sclerosis.

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS.

Self-Reported Medication Use and Urinary Drug Metabolites in the German Chronic Kidney Disease (GCKD) Study.

BACKGROUND: Polypharmacy is common among patients with CKD, but little is known about the urinary excretion of many drugs and their metabolites among patients with CKD. METHODS: To evaluate self-reported medication use in relation to urine drug metabolite levels in a large cohort of patients with CKD, the German Chronic Kidney Disease study, we ascertained self-reported use of 158 substances and 41 medication groups, and coded active ingredients according to the Anatomical Therapeutic Chemical Classification System. We used a nontargeted mass spectrometry-based approach to quantify metabolites in urine; calculated specificity, sensitivity, and accuracy of medication use and corresponding metabolite measurements; and used multivariable regression models to evaluate associations and prescription patterns. RESULTS: Among 4885 participants, there were 108 medication-drug metabolite pairs on the basis of reported medication use and 78 drug metabolites. Accuracy was excellent for measurements of 36 individual substances in which the unchanged drug was measured in urine (median, 98.5%; range, 61.1%-100%). For 66 pairs of substances and their related drug metabolites, median measurement-based specificity and sensitivity were 99.2% (range, 84.0%-100%) and 71.7% (range, 1.2%-100%), respectively. Commonly prescribed medications for hypertension and cardiovascular risk reduction-including angiotensin II receptor blockers, calcium channel blockers, and metoprolol-showed high sensitivity and specificity. Although self-reported use of prescribed analgesics (acetaminophen, ibuprofen) was <3% each, drug metabolite levels indicated higher usage (acetaminophen, 10%-26%; ibuprofen, 10%-18%). CONCLUSIONS: This comprehensive screen of associations between urine drug metabolite levels and self-reported medication use supports the use of pharmacometabolomics to assess medication adherence and prescription patterns in persons with CKD, and indicates under-reported use of medications available over the counter, such as analgesics.

Cytokine-specific autoantibodies shape the gut microbiome in autoimmune polyendocrine syndrome type 1.

BACKGROUND: Gastrointestinal dysfunction is a frequent and disabling manifestation of autoimmune polyendocrine syndrome type 1 (APS-1), a rare monogenic multiorgan autoimmune disease caused by the loss of central AIRE-controlled immune tolerance. OBJECTIVES: This study aimed to understand the role of the gut microbiome in APS-1 symptoms and potentially alleviate common gastrointestinal symptoms by probiotic intervention. METHODS: This study characterized the fecal microbiomes of 28 patients with APS-1 and searched for associations with gastrointestinal symptoms, circulating anti-cytokine autoantibodies, and tryptophan-related metabolites. Additionally, daily doses of the probiotic Lactobacillus rhamnosus GG were administered for 3 months. RESULTS: Of 581 metagenomic operational taxonomic units (mOTUs) characterized in total, 14 were significantly associated with patients with APS-1 compared with healthy controls, with 6 mOTUs depleted and 8 enriched in patients with APS-1. Four overabundant mOTUs were significantly associated with severity of constipation. Phylogenetically conserved microbial associations with autoantibodies against cytokines were observed. After the 3-month intervention with the probiotic L rhamnosus GG, a subset of gastrointestinal symptoms were alleviated. L rhamnosus GG abundance was increased postintervention and corresponded with decreased abundances of Alistipes onderdonkii and Collinsella aerofaciens, 2 species positively associated with severity of diarrhea in patients with APS-1. CONCLUSIONS: The APS-1 microbiome correlates with several APS-1 symptoms, some of which are alleviated after a 3-month L rhamnosus GG intervention. Autoantibodies against cytokines appear to shape the gut microbiome by positively correlating with a taxonomically consistent group of bacteria.

LDHB Overexpression Can Partially Overcome T Cell Inhibition by Lactic Acid.

Accelerated glycolysis leads to secretion and accumulation of lactate and protons in the tumor environment and determines the efficacy of adoptive T cell and checkpoint inhibition therapy. Here, we analyzed effects of lactic acid on different human CD4 T cell subsets and aimed to increase CD4 T cell resistance towards lactic acid. In all CD4 T cell subsets analyzed, lactic acid inhibited metabolic activity (glycolysis and respiration), cytokine secretion, and cell proliferation. Overexpression of the lactate-metabolizing isoenzyme LDHB increased cell respiration and mitigated lactic acid effects on intracellular cytokine production. Strikingly, LDHB-overexpressing cells preferentially migrated into HCT116 tumor spheroids and displayed higher expression of cytotoxic effector molecules. We conclude, that LDHB overexpression might be a promising strategy to increase the efficacy of adoptive T cell transfer therapy.

Metabolic Heterogeneity of Brain Tumor Cells of Proneural and Mesenchymal Origin.

Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. BTICs of different subtypes were challenged with oxidative phosphorylation (OXPHOS) inhibition with metformin to assess the differential effects of metabolic intervention on key resistance features. Whereas mesenchymal BTICs varied according to their invasiveness, they were in general more glycolytic and less responsive to metformin. Proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathway, and responded better to metformin. Targeting glycolysis may be a promising approach to inhibit tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors.

Urine Metabolite Levels, Adverse Kidney Outcomes, and Mortality in CKD Patients: A Metabolome-wide Association Study.

RATIONALE & OBJECTIVE: Mechanisms underlying the variable course of disease progression in patients with chronic kidney disease (CKD) are incompletely understood. The aim of this study was to identify novel biomarkers of adverse kidney outcomes and overall mortality, which may offer insights into pathophysiologic mechanisms. STUDY DESIGN: Metabolome-wide association study. SETTING & PARTICIPANTS: 5,087 patients with CKD enrolled in the observational German Chronic Kidney Disease Study. EXPOSURES: Measurements of 1,487 metabolites in urine. OUTCOMES: End points of interest were time to kidney failure (KF), a combined end point of KF and acute kidney injury (KF+AKI), and overall mortality. ANALYTICAL APPROACH: Statistical analysis was based on a discovery-replication design (ratio 2:1) and multivariable-adjusted Cox regression models. RESULTS: After a median follow-up of 4 years, 362 patients died, 241 experienced KF, and 382 experienced KF+AKI. Overall, we identified 55 urine metabolites whose levels were significantly associated with adverse kidney outcomes and/or mortality. Higher levels of C-glycosyltryptophan were consistently associated with all 3 main end points (hazard ratios of 1.43 [95% CI, 1.27-1.61] for KF, 1.40 [95% CI, 1.27-1.55] for KF+AKI, and 1.47 [95% CI, 1.33-1.63] for death). Metabolites belonging to the phosphatidylcholine pathway showed significant enrichment. Members of this pathway contributed to the improvement of the prediction performance for KF observed when multiple metabolites were added to the well-established Kidney Failure Risk Equation. LIMITATIONS: Findings among patients of European ancestry with CKD may not be generalizable to the general population. CONCLUSIONS: Our comprehensive screen of the association between urine metabolite levels and adverse kidney outcomes and mortality identifies metabolites that predict KF and represents a valuable resource for future studies of biomarkers of CKD progression.

LEF1 supports metastatic brain colonization by regulating glutathione metabolism and increasing ROS resistance in breast cancer.

More than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer-binding factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma.

Acquired resistance to DZNep-mediated apoptosis is associated with copy number gains of AHCY in a B-cell lymphoma model.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is considered an important driver of tumor development and progression by its histone modifying capabilities. Inhibition of EZH2 activity is thought to be a potent treatment option for eligible cancer patients with an aberrant EZH2 expression profile, thus the indirect EZH2 inhibitor 3-Deazaneplanocin A (DZNep) is currently under evaluation for its clinical utility. Although DZNep blocks proliferation and induces apoptosis in different tumor types including lymphomas, acquired resistance to DZNep may limit its clinical application. METHODS: To investigate possible mechanisms of acquired DZNep resistance in B-cell lymphomas, we generated a DZNep-resistant clone from a previously DZNep-sensitive B-cell lymphoma cell line by long-term treatment with increasing concentrations of DZNep (ranging from 200 to 2000 nM) and compared the molecular profiles of resistant and wild-type clones. This comparison was done using molecular techniques such as flow cytometry, copy number variation assay (OncoScan and TaqMan assays), fluorescence in situ hybridization, Western blot, immunohistochemistry and metabolomics analysis. RESULTS: Whole exome sequencing did not indicate the acquisition of biologically meaningful single nucleotide variants. Analysis of copy number alterations, however, demonstrated among other acquired imbalances an amplification (about 30 times) of the S-adenosyl-L-homocysteine hydrolase (AHCY) gene in the resistant clone. AHCY is a direct target of DZNep and is critically involved in the biological methylation process, where it catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine. The amplification of the AHCY gene is paralleled by strong overexpression of AHCY at both the transcriptional and protein level, and persists upon culturing the resistant clone in a DZNep-free medium. CONCLUSIONS: This study reveals one possible molecular mechanism how B-cell lymphomas can acquire resistance to DZNep, and proposes AHCY as a potential biomarker for investigation during the administration of EZH2-targeted therapy with DZNep.

A Novel Metabolic Signature To Predict the Requirement of Dialysis or Renal Transplantation in Patients with Chronic Kidney Disease.

Identification of chronic kidney disease patients at risk of progressing to end-stage renal disease (ESRD) is essential for treatment decision-making and clinical trial design. Here, we explored whether proton nuclear magnetic resonance (NMR) spectroscopy of blood plasma improves the currently best performing kidney failure risk equation, the so-called Tangri score. Our study cohort comprised 4640 participants from the German Chronic Kidney Disease (GCKD) study, of whom 185 (3.99%) progressed over a mean observation time of 3.70 ± 0.88 years to ESRD requiring either dialysis or transplantation. The original four-variable Tangri risk equation yielded a C statistic of 0.863 (95% CI, 0.831-0.900). Upon inclusion of NMR features by state-of-the-art machine learning methods, the C statistic improved to 0.875 (95% CI, 0.850-0.911), thereby outperforming the Tangri score in 94 out of 100 subsampling rounds. Of the 24 NMR features included in the model, creatinine, high-density lipoprotein, valine, acetyl groups of glycoproteins, and Ca2+-EDTA carried the highest weights. In conclusion, proton NMR-based plasma fingerprinting improved markedly the detection of patients at risk of developing ESRD, thus enabling enhanced patient treatment.

Double genetic disruption of lactate dehydrogenases A and B is required to ablate the "Warburg effect" restricting tumor growth to oxidative metabolism.

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout (LDHA/B-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B-DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the LDHA/B-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both LDHA and LDHB need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by LDHA/B genetic disruptions is responsible for the tumors' escape and growth.