Glycolytic Lactate Production Research Articles

Consequences of a 2-Deoxyglucose Exposure on the ATP Content and the Cytosolic Glucose Metabolism of Cultured Primary Rat Astrocytes

The glucose analogue 2-deoxyglucose (2DG) has frequently been used as a tool to study cellular glucose uptake and to inhibit glycolysis. Exposure of primary cultured astrocytes to 2DG caused a time- and concentration-dependent cellular accumulation of 2-deoxyglucose-6-phosphate (2DG6P) that was accompanied by a rapid initial decline in cellular ATP content. Inhibitors of mitochondrial respiration as well as inhibitors of mitochondrial uptake of pyruvate and activated fatty acids accelerated the ATP loss, demonstrating that mitochondrial ATP regeneration contributes to the partial maintenance of the ATP content in 2DG-treated astrocytes. After a 30 min exposure to 10 mM 2DG the specific content of cellular 2DG6P had accumulated to around 150 nmol/mg, while cellular ATP was lowered by 50% to around 16 nmol/mg. Following such a 2DG6P-loading of astrocytes, glycolytic lactate production from applied glucose was severely impaired during the initial 60 min of incubation, but was reestablished during longer incubation concomitant with a loss in cellular 2DG6P content. In contrast to glycolysis, the glucose-dependent NADPH regeneration via the pentose phosphate pathway (PPP) was only weakly affected in 2DG6P-loaded astrocytes and in cells that were coincubated with glucose in the presence of an excess of 2DG. Additionally, in the presence of 2DG PPP-dependent WST1 reduction was found to have doubled compared to hexose-free control incubations, indicating that cellular 2DG6P can serve as substrate for NADPH regeneration by the astrocytic PPP. The data presented provide new insights on the metabolic consequences of a 2DG exposure on the energy and glucose metabolism of astrocytes and demonstrate the reversibility of the inhibitory potential of a 2DG-treatment on the glucose metabolism of cultured astrocytes.

A BMP-controlled metabolic/epigenetic signaling cascade directs midfacial morphogenesis.

Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients and are associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that bone morphogenetic protein (BMP) signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53-dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling-dependent lactate generation drove histone lactylation levels to alter essential genes of Pdgfra, thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein BMP signaling controls a metabolic/epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation.

Abstract 3585: Probing the relationship between renal carcinoma perfusion, hypoxia, and metabolism during response to VEGFR inhibition and at resistance

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common primary kidney cancer. VEGF-receptor tyrosine kinase inhibitors remain a second-line or concurrent (with immunotherapy) treatment option for advanced RCC, but resistance inevitably occurs. There remains a need for biomarkers predictive of response to VEGFR targeting. Anti-VEGFR therapy-induced hypoxia can result in changes to both tumor metabolism and vascularization that contribute to the aggressiveness of treatment-resistant tumors. In this study we used in vivo hyperpolarized 13C-tert-butanol, a freely-diffusible novel MRI tracer that provides high-SNR tissue perfusion mapping, and hyperpolarized 13C pyruvate MRI, which enables real-time investigation of glycolytic lactate production, to understand the evolution of RCC perfusion and metabolism in response to VEGFR targeting, while correlating to histologic measures of tumor proliferation (Ki67) and vascularization (CD34). A498, a human ccRCC cell line, was implanted subcutaneously into 12-week-old nude mice. When tumors reached 15mm, the mice were placed into three arms, untreated (UT), sunitinib sensitive (SS), and sunitinib resistant (SR). SS mice were treated with sunitinib (25 mg/kg via gavage) then the mice were imaged with MRI at 48 hours. SR mice were treated with sunitinib daily until 2mm of re-growth was detected, then MRI was performed. UT mice were imaged when tumors reached 20mm. Pimonidazole infusion immediately after MRI, then the tumors were harvested. Histology included Ki67, CD34, and pimonidazole (hypoxia) staining. Ki67 expression was greater in the UT group compared to SS (37.56±5.21 vs 26.23±3.78, p=0.004), but not compared to SR (37.56±5.21 vs 34.32±1.12, p=0.93). The UT group demonstrated greater vascularization with CD-34 vessel counting than SS (UT 6.70±1.81 vs. SS 2.39±1.16, p=0.002), while vascularization was increased in SR compared to SS (SR 3.82±1.81 vs SS 2.39±1.16, p=0.05). Ki67 expression was positively correlated with CD34 expression. MR-measured tumor perfusion of the SS group was greater than UT (0.0086±0.0056 vs 0.0035±0.0026, p=0.0001) and SR (0.0086±0.0056 vs 0.0043±0.0033, p=0.0003). Tumor lactate production in the SS group was lower than UT (0.752±0.268 vs 0.609±0.188, p=0.04) and SR (0.752±0.268 vs 0.593±0.246, p=0.006). However, tumor perfusion was negatively correlated with lactate production ratio only in the SR group. Untreated and sunitinib-resistent RCC tumors are associated with increased perfusion and vascularity, and decreased lactate production. An inverse relationship between lactate production and perfusion in SR may be attributable to vascular normalization. Analysis of the spatial relationship between tumor metabolism and perfusion will further yield additional detail on the interplay between hypoxia and metabolism. Citation Format: Qianhui Dou, Patricia Coutinho de Souza, Xiaoen Wang, Aaron K. Grant, Leo L. Tsai. Probing the relationship between renal carcinoma perfusion, hypoxia, and metabolism during response to VEGFR inhibition and at resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3585.

Glycolysis-related lncRNA TMEM105 upregulates LDHA to facilitate breast cancer liver metastasis via sponging miR-1208

Increased glycolysis is one of the key metabolic hallmarks of cancer cells. However, the roles of lncRNAs in energy metabolism and cancer metastasis remain unclear. Here, the expression of TMEM105 associated with glycolysis was dramatically elevated from normal to breast cancer to breast cancer liver metastasis tissues, and the survival analysis revealed that high TMEM105 expression was related to poor survival, especially in patients with liver metastasis. Moreover, TMEM105 facilitated the glycolysis of breast cancer cells and induced cell invasion and breast cancer liver metastasis (BCLM). Mechanistically, TMEM105 regulated LDHA expression by sponging miR-1208, which further promoted cell glycolysis and BCLM. Importantly, glycolytic production of lactate enhanced TMEM105 expression in breast cancer cells by activating the SHH-MAZ signaling pathway. These findings suggested that the lactate-responsive TMEM105 acted as a miRNA sponge, inducing BCLM via a glycolysis-mediated positive feedback loop, which might be a rational target for the treatment of BCLM patients.

BIOM-10. PRECLINICAL PLATFORM FOR THE IDENTIFICATION OF DEUTERIUM MAGNETIC RESONANCE SPECTROSCOPY-BASED BIOMARKERS OF BRAIN TUMOR METABOLISM

Abstract Metabolic reprogramming is a fundamental hallmark of cancer, which can be exploited for non-invasive tumor imaging. Deuterium magnetic resonance spectroscopy (2H-MRS) recently emerged as a novel, translational method of interrogating flux from 2H-labeled substrates to metabolic products. However, to date, preclinical studies have been performed in vivo, an endeavor which suffers from low-throughput and potential wastage of animal life, especially when considering studies of treatment response. Developing in vitro assays for monitoring metabolism of 2H-labeled substrates will enhance throughput, lead to the rapid evaluation of new 2H-based probes and enable identification of treatment response biomarkers, thereby allowing the best 2H-based probes to be translated for further in vivo assessment. The goal of this study was to develop a preclinical cell-based platform for quantifying metabolism of 2H-labeled probes in brain tumor models. Since the Warburg effect, which is characterized by elevated glycolytic production of lactate, is a metabolic phenotype of cancer, including brain tumors, we examined metabolism of 2H-glucose or 2H-pyruvate in patient-derived glioblastoma (GBM6) and oligodendroglioma (BT88) cells and compared to normal human astrocytes (NHACONTROL). Following incubation in media containing [6,6’-2H]glucose or [U-2H]pyruvate, 2H-MR spectra obtained from live cell suspensions showed elevated 2H-lactate production in GBM6 and BT88 cells relative to NHACONTROL. Importantly, 2H-lactate production from [6,6’-2H]glucose or from [U-2H]pyruvate was reduced in GBM6 or BT88 cells subjected to irradiation and temozolomide, which is standard of care for glioma patients, pointing to the utility of this method for detecting response to therapy. Collectively, we have, for the first time, demonstrated the ability to quantify metabolism of 2H-MRS probes in live cell suspensions and validated the utility of our assay for differentiating tumor from normal cells and assessing response to therapy. Our studies will expedite the identification of novel 2H-MRS probes for imaging brain tumors and potentially other types of cancer.

Lemon myrtle extract inhibits lactate production by Streptococcus mutans.

Backhousia citriodora (lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans. In addition to glucansucrase, various virulence factors in S. mutans are involved in the initiation of caries. Lactate produced by S. mutans demineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutans lactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutans culture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutans cell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay.

Altered skeletal muscle metabolic pathways, age, systemic inflammation, and low cardiorespiratory fitness associate with improvements in disease activity following high-intensity interval training in persons with rheumatoid arthritis

BackgroundExercise training, including high-intensity interval training (HIIT), improves rheumatoid arthritis (RA) inflammatory disease activity via unclear mechanisms. Because exercise requires skeletal muscle, skeletal muscle molecular pathways may contribute. The purpose of this study was to identify connections between skeletal muscle molecular pathways, RA disease activity, and RA disease activity improvements following HIIT.MethodsRA disease activity assessments and vastus lateralis skeletal muscle biopsies were performed in two separate cohorts of persons with established, seropositive, and/or erosive RA. Body composition and objective physical activity assessments were also performed in both the cross-sectional cohort and the longitudinal group before and after 10 weeks of HIIT. Baseline clinical assessments and muscle RNA gene expression were correlated with RA disease activity score in 28 joints (DAS-28) and DAS-28 improvements following HIIT. Skeletal muscle gene expression changes with HIIT were evaluated using analysis of covariance and biological pathway analysis.ResultsRA inflammatory disease activity was associated with greater amounts of intramuscular adiposity and less vigorous aerobic exercise (both p < 0.05). HIIT-induced disease activity improvements were greatest in those with an older age, elevated erythrocyte sedimentation rate, low cardiorespiratory fitness, and a skeletal muscle molecular profile indicative of altered metabolic pathways (p < 0.05 for all). Specifically, disease activity improvements were linked to baseline expression of RA skeletal muscle genes with cellular functions to (1) increase amino acid catabolism and interconversion (GLDC, BCKDHB, AASS, PYCR, RPL15), (2) increase glycolytic lactate production (AGL, PDK2, LDHB, HIF1A), and (3) reduce oxidative metabolism via altered beta-oxidation (PXMP2, ACSS2), TCA cycle flux (OGDH, SUCLA2, MDH1B), and electron transport chain complex I function (NDUFV3). The muscle mitochondrial glycine cleavage system (GCS) was identified as critically involved in RA disease activity improvements given upregulation of multiple GCS genes at baseline, while GLDC was significantly downregulated following HIIT.ConclusionIn the absence of physical activity, RA inflammatory disease activity is associated with transcriptional remodeling of skeletal muscle metabolism. Following exercise training, the greatest improvements in disease activity occur in older, more inflamed, and less fit persons with RA. These exercise training-induced immunomodulatory changes may occur via reprogramming muscle bioenergetic and amino acid/protein homeostatic pathways.Trial registrationClinicalTrials.gov, NCT02528344. Registered on 19 August 2015.

Abstract 2403: Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer

Abstract Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to current treatment regimens contributes to the dismal prognosis for patients diagnosed with the disease. Accumulated evidence indicates that metabolic alterations may play a role in chemo-resistance. In this project, we investigated the role of pyruvate dehydrogenase Alpha1 (PDHA1) in chemo-resistance. Methods: Wild type and Gemcitabine-resistant pancreatic cancer cell lines MIA PaCa-2, HPAF-II, and UN-KPC-961 were used. Mitochondrial respiration, ATP production and glycolytic activities were measured by seahorse. PDHA1 was overexpressed or its activity inhibited by over-expressing PDHA1 kinase in these cells and cancer cell survival was measured by MTT and cancer stemness measured by Western and RT-PCR. PDHA1 level was measured in human and mouse PDAC tissues. Results: Seahorse analysis showed a significantly lower mitochondrial activity, ATP production, and increased glycolytic activity and lactate production in gemcitabine-resistant compared to non- resistant pancreatic cancer cells. PDHA1 mRNA and protein levels were less in gemcitabine-resistant cancer cells compared to non-resistant cells. PDHA1 levels were less in human PDAC tissues from patients and mice who developed resistance to chemotherapy. PDHA1 over-expression in gemcitabine-resistant cancer cells reversed resistance to chemotherapy, decreased cancer cell survival and decreased expression of cancer stemness markers. Whereas, PDHA1 inhibition by over-expressing PDHA1 kinase, in non-resistant cancer cells, induced resistance to chemotherapy, increased cancer cell survival and increased expression of cancer stemness markers. We found that inhibition of histone deacetylase (HDAC) (class 1 and 2) increased transcription level of PDHA1 and reversed chemo-resistance. Conclusion: We show that PDHA1 is a regulator of chemo-resistance in pancreatic cancer cells. PDHA1is a novel target for treating chemo-resistant PDAC. Citation Format: Fouzia Zayou, Chintan Chheda, Adrian Lim, Omer H. Elmadbouh, Stephen J. Pandol, Mouad Edderkaoui. Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2403.

Abstract 233: Tumor-associated carbonic anhydrase IX maintains cellular proliferation by regulating tumor metabolism: a novel link revealed by proteomics

Abstract Tumor hypoxia is a critical microenvironmental stress occurring in virtually all types of solid tumors. Survival of cancer cells is dependent upon their ability to adapt to these limiting conditions. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a crucial adaptive role in response to hypoxic and acidic microenvironments by catalytically hydrating extracellular CO2 to produce bicarbonate ions that maintain intracellular pH (pHi). In order to determine how CAIX function impacts other cellular processes, we used proteomic profiling to identify changes in response to transient CA IX knockdown in hypoxia. We discovered a significant reduction of key glycolytic enzyme levels with a most prominent decrease in lactate dehydrogenase (LDH). Our data shows that in addition to participating in cellular regulation of pHi, CA IX significantly stimulates glycolytic flux, lactate production and rate of proliferation in hypoxia. Because LDH activity contributes to pHi regulation by consuming cytoplasmic protons in the process of NADH regeneration, CA IX knockdown-mediated LDH suppression may further compromise the pHi regulating ability in hypoxia. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate compensates for reduced lactate production and restores pHi and proliferation in CA IX-deficient cells. These findings suggest that CA IX confers adaptive plasticity and supports proliferation of cancer cells both directly and indirectly - through its effect as a pHi regulator and by maintaining glycolysis-permissive intracellular environment, respectively. Because CA IX expression is limited almost exclusively to tumors, our findings highlight the role as well as provide mechanistic insight for the utility of CA IX as an anti-cancer target and suggests that highly glycolytic tumors might be particularly vulnerable to CA IX-targeted therapy. Citation Format: Martin Benej, Eliska Svastova, Radivojka Banova, Juraj Kopacek, Adriana Gibadulinova, Martin Kery, Simona Arena, Andrea Scaloni, Monica Vitale, Nicola Zambrano, Ioanna Papandreou, Nicholas C. Denko, Silvia Pastorekova. Tumor-associated carbonic anhydrase IX maintains cellular proliferation by regulating tumor metabolism: a novel link revealed by proteomics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 233.

Glucose metabolism links astroglial mitochondria to cannabinoid effects.

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1-5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5-7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.

β1-adrenoceptor-stimulated lactate production in cultured astrocytes is predominantly glycogen-independent

Noradrenaline (NA) promotes breakdown of the glucose-polymer, glycogen, and hence enhances glycolytic production of lactate in astrocytes. Here, in cultured rat cerebrocortical astrocytes, we examined the contributions of different adrenoceptor subtypes to NA-modulated glucose metabolism, and the relationship of NA-induced glycogenolysis to lactate production. Stimulation of astrocytic glucose metabolism by NA was mediated predominantly via β1-adrenoceptors and cAMP. Constitutive β 1-adrenoceptor activity – in the absence of exogenous NA - contributed to the basal rate of glycogen turnover. Although mRNAs encoding both β 1- and β 2-adrenoceptors were detected in these astrocytes, β 2-adrenoceptors contributed little to NA-induced modulation of glucose metabolism. Activation of α2- and α 1-adrenoceptors in these cells decreased cAMP and increased cytosolic Ca2+, respectively, but did not modulate NA-induced glycogenolysis: α 2-adrenoceptors because glycogenolysis was induced maximally by NA concentrations that only began to inhibit cAMP production; and α 1-adrenoceptors possibly because of desensitisation and depletion of Ca2+ stores. Under basal conditions, astrocytes converted glucose to extracellular lactate in near stoichiometric manner. When glucose-starved astrocytes were given fresh glucose-containing medium, lactate accumulation displayed a brief lag period before attaining a steady-state rate. During this lag period NA, acting at β 1-adrenoceptors, increased the rate of lactate accumulation both in the absence and presence of an inhibitor of glycogen turnover. At the steady-state, the rate of glucose incorporation into accumulated glycogen was ~ 5% of that into lactate, but NA enhanced lactate output by 20–50%: this further indicates that NA, via β 1-adrenoceptors and cAMP, can enhance astrocytic lactate production independently of its effect on glycogen turnover.

Barth Syndrome: Exploring Cardiac Metabolism with Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Barth syndrome (BTHS) is an X-linked recessive multisystem disorder caused by mutations in the TAZ gene (TAZ, G 4.5, OMIM 300394) that encodes for the acyltransferase tafazzin. This protein is highly expressed in the heart and plays a significant role in cardiolipin biosynthesis. Heart disease is the major clinical manifestation of BTHS with a high incidence in early life. Although the genetic basis of BTHS and tetralinoleoyl cardiolipin deficiency in BTHS-affected individuals are well-established, downstream metabolic changes in cardiac metabolism are still uncovered. Our study aimed to characterize TAZ-induced metabolic perturbations in the heart. Control (PGP1-TAZWT) and TAZ mutant (PGP1-TAZ517delG) iPS-CM were incubated with 13C6-glucose and 13C5-glutamine and incorporation of 13C into downstream Krebs cycle intermediates was traced. Our data reveal that TAZ517delG induces accumulation of cellular long chain acylcarnitines and overexpression of fatty acid binding protein (FABP4). We also demonstrate that TAZ517delG induces metabolic alterations in pathways related to energy production as reflected by high glucose uptake, an increase in glycolytic lactate production and a decrease in palmitate uptake. Moreover, despite mitochondrial dysfunction, in the absence of glucose and fatty acids, TAZ517delG-iPS-CM can use glutamine as a carbon source to replenish the Krebs cycle.

Metabolic vulnerabilities in IDH1/2‐mutated solid tumours lead to phase IB/II clinical trial using metformin and chloroquine

IntroductionHotspot mutations in the genes IDH1 and IDH2 (isocitrate dehydrogenase 1 and 2) occur in 60% of chondrosarcoma, 80% of WHO grade II–IV glioma and 20% of intrahepatic cholangiocarcinoma. Mutations induce a neoenzymatic activity that leads to the production and accumulation of D‐2‐hydroxyglutarate (D‐2HG). This causes metabolic rewiring and stress that is not fully understood. We previously postulated that the tricarboxylic acid (TCA) cycle, rather than glycolytic lactate production, is the predominant metabolic pathway in IDH1MUT glioma.ResultsTo confirm this hypothesis in a clinical setting, we used a novel technique of quantitative targeted next‐generation sequencing of 104 metabolic enzymes, on a cohort of 66 clinical gliomas. We confirmed the predominance of the oxidative metabolism in IDH1MUT gliomas, which is driven by lactate and glutamate anaplerosis. Preferential glutamate processing in IDH1MUT gliomas was confirmed by in situ enzyme activity mapping experiments in clinical gliomas of known IDH status, indicating that IDH1MUT gliomas depend on glutamatolysis, rather than glutaminolysis. We aim to specifically inhibit the metabolic processes that are essential to IDHMUT tumours using the oral antidiabetic metformin and the oral antimalarial drug chloroquine, which specifically target the metabolic vulnerabilities that are caused by IDHMUT.Clinical trialWe started a dose‐finding phase Ib/II clinical trial, in which patients with IDHMUT chondrosarcoma, glioma and intrahepatic cholangiocarcinoma are treated with a combination of metformin and chloroquine. The primary objective is to determine the maximum tolerated dose to establish the recommended dose for a phase II clinical trial. Secondary objectives of the study include (1) determination of pharmacokinetics and toxic effects of the study therapy, (2) investigation of tumour responses to metformin plus chloroquine in IDHMUT cancers using CT/MRI scans; and (3) whether tumour responses can be measured by noninvasive D‐2HG measurements (mass spectrometry and magnetic resonance spectroscopy) of tumour tissue, serum, urine, and/or bile or next‐generation sequencing of circulating tumour DNA (liquid biopsies). This study may open a novel treatment avenue for IDHMUT high‐grade chondrosarcoma, glioma and intrahepatic cholangiocarcinoma by repurposing the combination of two inexpensive drugs that are already approved for other indications.Support or Funding InformationThis work was supported by Dutch Cancer Society Grants KWF‐UVA 2014‐6839 (to M.K., R.J.M., and C.J.F.V.N.) and AMC2016.1‐10460 (to M.K., R.J.M., J.W.W., and C.J.F.V.N.). A) Targeted RNAseq of clinical glioma samples. Gene expression levels of genes involved in glycolysis, glutaminolysis and the TCA cycle. B) Metabolic mapping of glutamate dehydrogenase (GLUD) and glutaminase (GLS). Cryostat sections and (C) quantification activity levels in IDH1WT (n=5) and IDH1MUT (n=7)imageA) Targeted RNAseq of clinical glioma samples. Gene expression levels of genes involved in glycolysis, glutaminolysis and the TCA cycle. B) Metabolic mapping of glutamate dehydrogenase (GLUD) and glutaminase (GLS). Cryostat sections and (C) quantification activity levels in IDH1WT (n=5) and IDH1MUT (n=7) D) Cellular carbohydrate and glutamine metabolism showing the pathways in which IDH1 and IDH2 are functional. The conversion of glutamine into α‐ketoglutarate occurs in the glutaminolysis pathway and the last step is catalysed by glutamate dehydrogenase (GLUD), which is inhibited by chloroquine and metformin. Complex I of the electron transport chain (ETC) is also inhibited by metformin. E) Dosing schedule and study design for patients. MRS, magnetic resonance spectroscopy; MS, mass spectrometryimageD) Cellular carbohydrate and glutamine metabolism showing the pathways in which IDH1 and IDH2 are functional. The conversion of glutamine into α‐ketoglutarate occurs in the glutaminolysis pathway and the last step is catalysed by glutamate dehydrogenase (GLUD), which is inhibited by chloroquine and metformin. Complex I of the electron transport chain (ETC) is also inhibited by metformin. E) Dosing schedule and study design for patients. MRS, magnetic resonance spectroscopy; MS, mass spectrometryThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exposure of Cultured Astrocytes to Menadione Triggers Rapid Radical Formation, Glutathione Oxidation and Mrp1-Mediated Export of Glutathione Disulfide.

Menadione (2-methyl-1,4-naphthoquinone) is a synthetic derivative of vitamin K that allows rapid redox cycling in cells and thereby generates reactive oxygen species (ROS). To test for the consequences of a treatment of brain astrocytes with menadione, we incubated primary astrocyte cultures with this compound. Incubation with menadione in concentrations of up to 30µM did not affect cell viability. In contrast, exposure of astrocytes to 100µM menadione caused a time-dependent impairment of cellular metabolism and cell functions as demonstrated by impaired glycolytic lactate production and strong increases in the activity of extracellular lactate dehydrogenase and in the number of propidium iodide-positive cells within 4h of incubation. In addition, already 5min after exposure of astrocytes to menadione a concentration-dependent increase in the number of ROS-positive cells as well as a concentration-dependent and transient accumulation of cellular glutathione disulfide (GSSG) were observed. The rapid intracellular GSSG accumulation was followed by an export of GSSG that was prevented in the presence of MK571, an inhibitor of the multidrug resistance protein 1 (Mrp1). Menadione-induced glutathione (GSH) oxidation and ROS formation were found accelerated after glucose-deprivation, while the presence of dicoumarol, an inhibitor of the menadione-reducing enzyme NQO1, did not affect the menadione-dependent GSSG accumulation. Our study demonstrates that menadione rapidly depletes cultured astrocytes of GSH via ROS-induced oxidation to GSSG that is subsequently exported via Mrp1.

IDH1‐mutated gliomas rely on anaplerosis of glutamate and lactate whereas IDH1 wild‐type gliomas rely on glycolysis and acetate anaplerosis

IntroductionHotspot mutations in isocitrate dehydrogenase 1 (IDH1MUT) initiate low grade glioma (LGG) and secondary glioblastoma and induce neomorphic activity that converts α‐ketoglutarate (α‐KG) to the oncometabolite D‐2‐hydroxyglutarate (D‐2HG). This causes metabolic rewiring and stress that is not fully understood. In vitro studies suggest that this stress is relieved via glutaminolysis, providing cells with α‐KG via activities of glutaminase (GLS) and glutamate dehydrogenase (GLUD). We previously postulated that IDH1MUT gliomas directly use glutamate as a shortcut.ResultsTo test this hypothesis, we first showed by in silico analysis of 269 IDH1 wild‐type (IDH1WT) and 408 IDH1MUT gliomas, obtained from the The Cancer Genome Atlas (TCGA) database, that IDH1WT gliomas have high expression levels of genes encoding for enzymes that are involved in glycolysis and acetate anaplerosis. On the other hand, the tricarboxylic acid (TCA) cycle, rather than glycolytic lactate production, is the predominant metabolic pathway in IDH1MUT gliomas and is driven by lactate and glutamate anaplerosis to facilitate production of α‐KG, and ultimately D‐2‐HG. Secondly, we confirmed these findings by a novel technique of quantitative targeted next‐generation sequencing of RNAs encoding for 104 metabolic enzymes, on a cohort of 66 clinical gliomas. Preferential glutamate processing in IDH1MUT gliomas was confirmed by in situ enzyme activity mapping experiments in clinical gliomas of known IDH status. We functionally validated these findings in a set of glioma xenografts with and without the endogenous IDH1R132H mutation, indicating that IDH1MUT gliomas depend on glutamatolysis, rather than glutaminolysis. We show that transcript levels in our xenocraft models are in good agreement with our in silico analysis of the TCGA database. Finally, we confirmed the glutamate dependency of IDH1MUT gliomas by in vivo magnetic resonance spectroscopic imaging, whereas IDH1WT gliomas showed high lactate levels.ConclusionsWe show that IDH1WT gliomas have a typical Warburg phenotype and rely on acetate anaplerosis whereas IDH1MUT gliomas are glutamate and lactate dependent. This metabolic rewiring in IDH1MUT glioma, enables targeting of glutaminolysis rather than direct inhibition of IDH1MUT for therapy. It diminishes the supply of glutamate‐derived α‐KG and directly inhibits the production of D‐2HG and simultaneously worsen the redox status of the glioma cells by inhibiting NAD(P)H production by GLUD. A candidate drug to inhibit GLUD is epigallocatechin‐3‐gallate gallate, a component of green tea that is currently receiving high interest as anticancer agent.Support or Funding InformationThis work was supported by Dutch Cancer Society grant UvA 2014‐6839 (KL, MK) and by Stichting StopHersentumoren (CNAMvdH). RJM was supported by an AMC PhD scholarship. THP was supported by EFRO/GO project Ultrasense NMR.

Metformin Accelerates Glycolytic Lactate Production in Cultured Primary Cerebellar Granule Neurons

Metformin is the most frequently used drug for the treatment of type-II diabetes. As metformin has been reported to cross the blood-brain barrier, brain cells will encounter this drug. To test whether metformin may affect the metabolism of neurons, we exposed cultured rat cerebellar granule neurons to metformin. Treatment with metformin caused a time- and concentration-dependent increase in glycolytic lactate release from viable neurons as demonstrated by the three-to fivefold increase in extracellular lactate concentration determined after exposure to metformin. Half-maximal stimulation of lactate production was found after incubation of neurons for 4h with around 2mM or for 24h with around 0.5mM metformin. Neuronal cell viability was not affected by millimolar concentrations of metformin during acute incubations in the hour range nor during prolonged incubations, although alterations in cell morphology were observed during treatment with 10mM metformin for days. The acute stimulation of neuronal lactate release by metformin was persistent upon removal of metformin from the medium and was not affected by the presence of modulators of adenosine monophosphate activated kinase activity. In contrast, rabeprazole, an inhibitor of the organic cation transporter 3, completely prevented metformin-mediated stimulation of neuronal lactate production. In summary, the data presented identify metformin as a potent stimulator of glycolytic lactate production in viable cultured neurons and suggest that organic cation transporter 3 mediates the uptake of metformin into neurons.

Abstract 431: IDH1-mutated gliomas rely on anaplerosis of glutamate and lactate whereas IDH1 wild-type gliomas rely on glycolysis and acetate anaplerosis

Abstract Hotspot mutations in isocitrate dehydrogenase 1 (IDH1MT) initiate low grade glioma (LGG) and secondary glioblastoma and induce neomorphic activity that converts α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2-HG). This causes metabolic rewiring that is not fully understood and in vitro studies have shown that IDH1MT cancer cells rely on glutaminolysis, providing cells with α-KG via activities of glutaminase (GLS) and glutamate dehydrogenase (GLUD). Here, we first show by in silico analysis of 269 IDH1 wild-type (IDH1WT) and 408 IDH1MT gliomas, obtained from the The Cancer Genome Atlas (TCGA) database, that IDH1WT gliomas have high expression levels of genes encoding for enzymes that are involved in glycolysis and acetate anaplerosis. On the other hand, the tricarboxylic acid (TCA) cycle, rather than glycolytic lactate production, is the predominant metabolic pathway in IDH1MT gliomas and is driven by lactate and glutamate anaplerosis to facilitate production of α-KG, and ultimately D-2-HG. IDH1WT- and IDH1MT-related differences in expression were found in both LGG and glioblastoma. Furthermore, via in situ enzymatic activity mapping, we show in human gliomas and in xenocraft models that GLUD activity is increased and GLS activity is decreased in IDH1MT glioma, indicating that IDH1MT gliomas depend on glutamatolysis, rather than glutaminolysis. We show that transcript levels in our xenocraft models are in good agreement with our in silico analysis of the TCGA database. Finally, we confirmed the glutamate dependency of IDH1MT gliomas by MRS-flux analysis, whereas IDH1WT gliomas show high lactate production. Taken together, we show that IDH1WT gliomas have a typical Warburg phenotype and rely on acetate anaplerosis whereas IDH1MT gliomas are glutamate and lactate dependent. This metabolic rewiring in IDH1MT glioma, enables targeting of glutaminolysis rather than direct inhibition of IDH1MT for therapy. It diminishes the supply of glutamate-derived α-KG and directly inhibits the production of D-2-HG and simultaneously worsen the redox status of the glioma cells by inhibiting NAD(P)H production by GLUD. A candidate drug to inhibit GLUD is epigallocatechin-3-gallate gallate, a component of green tea that is currently receiving high interest as anti-cancer agent. Note: This abstract was not presented at the meeting. Citation Format: Mohammed Khurshed, Krissie Lenting, Remco J. Molenaar, William P. Leenders, Cornelis J. van Noorden. IDH1-mutated gliomas rely on anaplerosis of glutamate and lactate whereas IDH1 wild-type gliomas rely on glycolysis and acetate anaplerosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 431. doi:10.1158/1538-7445.AM2017-431

Abstract 4404: Novel approach to circumvent cisplatin resistant lung cancer by targeting metabolism

Abstract For the past three decades, there are no drugs which could reverse cisplatin resistance or selectively kill these resistant cells. We have discovered that cisplatin resistant (CR) lung cancer shares one common factor which is increase in reactive oxygen species (ROS). Decreased intracellular thioredoxin-1(TRX1) due to excessive secretion was found in vitro and in vivo, as well as in patients’ serum which could be a primary contributory factor to higher mitochondria-ROS levels. Furthermore, CR cells possess significantly increased number of mitochondria and consume higher rates of oxygen. Key glycolytic enzymes (HKII and LDHA) and lactate production were decreased in CR cells which suggests rewiring of tumor metabolic pathways. NAD+, crucial co-factor of all redox systems, is significantly reduced in all CR cells. Importantly, treatment with riluzole (the FDA approved drug that interferes with cystine/glutamate pump and results in reduced intracellular glutathione (GSH) levels) led to further decrease in NAD+ and LDHA, as well as heightened oxidative stress in CR cells. ROS levels were increased (2-3X) more in resistant cells after riluzole treatment while no significant change occurred in parental cells. The ID50 dosage of parental cells were 3-4 fold more than theirs CR cell counterparts and no cytotoxicity were found in normal lung fibroblast (n=8; p&amp;lt;0.05). Addition of 200 µM NAD+ re-stabilized LDHA, reduced ROS, and reversed riluzole induced cell death. To further determine whether LDHA correlates with ROS levels and sensitivity to riluzole, we inhibited LDHA expressions in parental cell lines S and H460 using siRNA. Lactate productions were decreased in knockdown cells and further attenuated upon riluzole treatment when compared with scrambled control. Higher basal levels of ROS were obtained from S+siLDHA and H460+siLDHA and further increased in ROS levels were observed when treated with riluzole. We then evaluated the viability of these cells under riluzole treatment. Both siLDHA clones were sensitive to riluzole particularly in S+siLDHA which showed 4 fold more sensitivity. Importantly, treatment of riluzole also led to further decrease in LDHA expressions at both protein and mRNA levels. Using riluzole in 2 mouse xenograft models (H460CR (KRAS mutant) and SC (wt)) resulted in completely disappeared in wild type mice and were significant reduced in KRAS mutant mice (n=5 per treatment group; p&amp;lt;0.002). Repurpose of riluzole should be considered for future treatment of CR lung cancer patients. Supported by Department of Veterans Affairs, CDA2 award (1K2BX001289) and Woman Cancer Association Fund. Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Lynn G. Feun, Macus T. Kuo, Niramol Savaraj. Novel approach to circumvent cisplatin resistant lung cancer by targeting metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2017-4404

In silico gene expression analysis reveals glycolysis and acetate anaplerosis in IDH1 wild-type glioma and lactate and glutamate anaplerosis in IDH1-mutated glioma.

Hotspot mutations in isocitrate dehydrogenase 1 (IDH1) initiate low-grade glioma and secondary glioblastoma and induce a neomorphic activity that converts α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2-HG). It causes metabolic rewiring that is not fully understood. We investigated the effects of IDH1 mutations (IDH1MUT) on expression of genes that encode for metabolic enzymes by data mining The Cancer Genome Atlas. We analyzed 112 IDH1 wild-type (IDH1WT) versus 399 IDH1MUT low-grade glioma and 157 IDH1WT versus 9 IDH1MUT glioblastoma samples. In both glioma types, IDH1WT was associated with high expression levels of genes encoding enzymes that are involved in glycolysis and acetate anaplerosis, whereas IDH1MUT glioma overexpress genes encoding enzymes that are involved in the oxidative tricarboxylic acid (TCA) cycle. In vitro, we observed that IDH1MUT cancer cells have a higher basal respiration compared to IDH1WT cancer cells and inhibition of the IDH1MUT shifts the metabolism by decreasing oxygen consumption and increasing glycolysis. Our findings indicate that IDH1WT glioma have a typical Warburg phenotype whereas in IDH1MUT glioma the TCA cycle, rather than glycolytic lactate production, is the predominant metabolic pathway. Our data further suggest that the TCA in IDH1MUT glioma is driven by lactate and glutamate anaplerosis to facilitate production of α-KG, and ultimately D-2-HG. This metabolic rewiring may be a basis for novel therapies for IDH1MUT and IDH1WT glioma.

Metabolic Response to Everolimus in Patient-Derived Triple-Negative Breast Cancer Xenografts.

Patients with triple-negative breast cancer (TNBC) are unresponsive to endocrine and anti-HER2 pharmacotherapy, limiting their therapeutic options to chemotherapy. TNBC is frequently associated with abnormalities in the PI3K/AKT/mTOR signaling pathway; drugs targeting this pathway are currently being evaluated in these patients. However, the response is variable, partly due to heterogeneity within TNBC, conferring a need to identify biomarkers predicting response and resistance to targeted therapy. In this study, we used a metabolomics approach to assess response to the mTOR inhibitor everolimus in a panel of TNBC patient-derived xenografts (PDX) (n = 103 animals). Tumor metabolic profiles were acquired using high-resolution magic angle spinning magnetic resonance spectroscopy. Partial least-squares-discriminant analysis on relative metabolite concentrations discriminated treated xenografts from untreated controls with an accuracy of 67% (p = 0.003). Multilevel linear mixed-effects models (LMM) indicated reduced glycolytic lactate production and glutaminolysis after treatment, consistent with PI3K/AKT/mTOR pathway inhibition. Although inherent metabolic heterogeneity between different PDX models seemed to hinder prediction of treatment response, the metabolic effects following treatment were more pronounced in responding xenografts compared to nonresponders. Additionally, the metabolic information predicted p53 mutation status, which may provide complementary insight into the interplay between PI3K signaling and other drivers of disease progression.