Inhibition Of Oxidative Phosphorylation Research Articles

Metabolic reprogramming toward oxidative phosphorylation identifies a therapeutic target for mantle cell lymphoma.

Metabolic reprogramming is linked to cancer cell growth and proliferation, metastasis, and therapeutic resistance in a multitude of cancers. Targeting dysregulated metabolic pathways to overcome resistance, an urgent clinical need in all relapsed/refractory cancers, remains difficult. Through genomic analyses of clinical specimens, we show that metabolic reprogramming toward oxidative phosphorylation (OXPHOS) and glutaminolysis is associated with therapeutic resistance to the Bruton's tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma (MCL), a B cell lymphoma subtype with poor clinical outcomes. Inhibition of OXPHOS with a clinically applicable small molecule, IACS-010759, which targets complex I of the mitochondrial electron transport chain, results in marked growth inhibition in vitro and in vivo in ibrutinib-resistant patient-derived cancer models. This work suggests that targeting metabolic pathways to subvert therapeutic resistance is a clinically viable approach to treat highly refractory malignancies.

BA6 Induces Apoptosis via Stimulation of Reactive Oxygen Species and Inhibition of Oxidative Phosphorylation in Human Lung Cancer Cells.

Lung cancer is the leading cause of cancer deaths in the world, with a five-year survival rate of less than 30%. Clinically effective chemotherapeutic treatments at the initial stage may eventually face the dilemma of no drug being effective due to drug resistance; therefore, finding new effective drugs for lung cancer treatment is a necessary and important issue. Compounds capable of further increasing the oxidative stress of cancer cells are considered to have anticancer potential because they possessed the ability to induce apoptosis. This study mainly investigated the effects of BA6 (heteronemin), the marine sponge sesterterpene, on lung cancer cell apoptosis, via modulation of mitochondrial reactive oxygen species (mtROS) and oxidative phosphorylation (OXPHOS). BA6 has cellular cytotoxic activities against a variety of cancer cell lines, but it has no effect on nontumor cells. The BA6-treated lung cancer cells show a significant increase in both cellular ROS and mtROS, which in turn caused the loss of mitochondrial membrane potential (MMP). The increase of oxidative stress in lung cancer cells treated with BA6 was accompanied by a decrease in the expression of antioxidant enzymes Cu/Zn SOD, MnSOD, and catalase. In addition, OXPHOS performed in the mitochondria and glycolysis in the cytoplasm were inhibited, which subsequently reduced downstream ATP production. Pretreatment with mitochondria-targeted antioxidant MitoTEMPO reduced BA6-induced apoptosis through the mitochondria-dependent apoptotic pathway, which was accompanied by increased cell viability, decreased mtROS, enhanced MMP, and suppressed expression of cleaved caspase-3 and caspase-9 proteins. In conclusion, the results of this study clarify the mechanism of BA6-induced apoptosis in lung cancer cells via the mitochondrial apoptotic pathway, suggesting that it is a potentially innovative alternative to the treatment of human lung cancer.

Metabolic Checkpoint of Immune Cells in Melanoma Brain Metastases.

Metabolic features of both cancer cells and immune cells shift with nutrient availability in the tumor microenvironment, resulting in differential effects on antitumor immune function. The work of Fischer and colleagues demonstrates that increased oxidative phosphorylation in brain metastases in patients with melanoma is a key regulator of intracranial immune surveillance and that inhibition of oxidative phosphorylation could reduce the incidence of intracranial brain metastases in a murine model of melanoma.See related article by Fischer et al., p. 628.

NLRX1 Facilitates HIV-1 Infection of CD4 T Cells by Promoting Oxidative Phosphorylation

Abstract The nucleotide-binding, leucine-rich-repeat-containing protein, NLRX1 is recognized as a host factor which promotes HIV-1 infection of myeloid cells by suppressing HIV-1 reverse transcribed DNA induced and STING-mediated innate immune response. However, the function of NLRX1 in HIV-1 infection of CD4 T cells, the major cells responsible for HIV-1 replication in vivo, is still unrevealed. Here we found NLRX1 still acted as a host factor required for HIV-1 infection of the human T cell line, primary CD4 T cells, and CD4 T cell-reconstituted humanized mice through a mechanism involved in cell intrinsic pathways but not innate immune cytokines. In the perspective of virology, we found NLRX1 facilitated both of the integration of HIV-1 cDNA and HIV-1 LTR-mediated viral gene transcription. By using SILAC-based quantitative proteomics, we characterized that HIV-1 infection-induced expression of genes involved in the electron transport chain (ETC) of mitochondria through an NLRX1 dependent mechanism. Consistent with the proteomics data, HIV-1 infection led to increased oxidative phosphorylation in Jurkat T cell line but not in the counterpart containing shRNAs against NLRX1. In addition, inhibition of oxidative phosphorylation by mitochondrial complex I inhibitors suppressed HIV-1 infection in Jurkat cells, while forcing expression of genes involved in ETC rescued the deficiency of HIV-1 replication in NLRX1-deficient cells. Importantly, treating humanized mice with FDA-approved drug, metformin which targets mitochondrial complex I, significantly reduced HIV-1 viral load. This study demonstrated the potential of targeting NLRX1 and applying metformin in suppressing HIV-1 replication in patients.

Molecular Profiling Reveals Unique Immune and Metabolic Features of Melanoma Brain Metastases.

There is a critical need to improve our understanding of the pathogenesis of melanoma brain metastases (MBM). Thus, we performed RNA sequencing on 88 resected MBMs and 42 patient-matched extracranial metastases; tumors with sufficient tissue also underwent whole-exome sequencing, T-cell receptor sequencing, and IHC. MBMs demonstrated heterogeneity of immune infiltrates that correlated with prior radiation and post-craniotomy survival. Comparison with patient-matched extracranial metastases identified significant immunosuppression and enrichment of oxidative phosphorylation (OXPHOS) in MBMs. Gene-expression analysis of intracranial and subcutaneous xenografts, and a spontaneous MBM model, confirmed increased OXPHOS gene expression in MBMs, which was also detected by direct metabolite profiling and [U-13C]-glucose tracing in vivo. IACS-010759, an OXPHOS inhibitor currently in early-phase clinical trials, improved survival of mice bearing MAPK inhibitor-resistant intracranial melanoma xenografts and inhibited MBM formation in the spontaneous MBM model. The results provide new insights into the pathogenesis and therapeutic resistance of MBMs. SIGNIFICANCE: Improving our understanding of the pathogenesis of MBMs will facilitate the rational development and prioritization of new therapeutic strategies. This study reports the most comprehensive molecular profiling of patient-matched MBMs and extracranial metastases to date. The data provide new insights into MBM biology and therapeutic resistance.See related commentary by Egelston and Margolin, p. 581.This article is highlighted in the In This Issue feature, p. 565.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Combination of Hypoglycemia and Metformin Impairs Tumor Metabolic Plasticity and Growth by Modulating the PP2A-GSK3β-MCL-1 Axis

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3β (GSK3β) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3β axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3β.

Targeting cancer cell metabolism in glioblastoma.

Parada and colleagues have identified a novel small-molecule inhibitor of oxidative phosphorylation, which exerts cancer-cell-specific toxicity and inhibits glioblastoma growth in mouse models.

Lethal Poisoning of Cancer Cells by Respiratory Chain Inhibition plus Dimethyl α-Ketoglutarate

Inhibition of oxidative phosphorylation (OXPHOS) by1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87), a complex I inhibitor, fails to kill human cancer cells invitro. Driven by this consideration, we attempted to identify agents that engage in synthetically lethal interactions with B87. Here, we report that dimethyl α-ketoglutarate (DMKG), a cell-permeable precursor of α-ketoglutarate that lacks toxicity on its own, kills cancer cells when combinedwith B87 or other inhibitors of OXPHOS. DMKG improved the antineoplastic effect of B87, both invitro and invivo. This combination caused MDM2-dependent, tumor suppressor protein p53 (TP53)-independent transcriptional reprogramming and alternative exon usage affecting multiple glycolytic enzymes, completely blocking glycolysis. Simultaneousinhibition of OXPHOS and glycolysis provokeda bioenergetic catastrophe culminating in the activation of a cell death program that involved disruption of the mitochondrial network and activation of PARP1, AIFM1, and APEX1. These results unveil a metabolic liability of human cancer cells that may be harnessed for the development of therapeutic regimens.

Platelet bioenergetics and its implications in disease

Bioenergetics drives energy production for mechanical processes that are critical to platelet functions, e.g., adhesion, spreading, aggregation, clot formation and contraction. Storage and pathological conditions e.g., stroke, sepsis, diabetes etc., alters platelet energetics and function; however, the specific mechanisms governing these alterations are not clear. It is not well understood which metabolic fuels can be used and which are required. We are investigating the roles of different metabolic pathways in the energy consuming process of clot retraction. Using a spectrum of techniques from in vitro platelet functional assays to biochemical methods, whole animal models, and measurement of bioenergetic functions of mitochondria we are elucidating platelet bioenergetics and its implications in disease. We developed a simple method for measuring clot contraction kinetics using time lapse imaging. Our system is efficient, high throughput and is favorable to study platelet bioenergetics of clot formation and retraction. We hypothesize that, upon activation with thrombin, fuel use can be highly plastic, depending on the fuels available. We found that glycolysis and oxidative phosphorylation can both contribute to provide energy for platelet function. Inhibitors of glycolysis and oxidative phosphorylation using 2‐Deoxyglucose and oligomycin inhibited clot retraction showing that both these processes are important.We have also begun to examine bioenergetics in platelets from diabetics. Platelets from type2 diabetes patients have been shown to have abnormal mitochondrial membrane potential, membrane fluidity, ATP content, and oxidative stress. However, the impact of diabetes on platelet bioenergetics is less clear. We have developed a mouse model to probe the role of mitochondria and oxidative phosphorylation in platelet function. TFAM (Mitochondrial Transcription factor A) regulates mitochondria DNA copy number and transcription of mitochondrial DNA encoded proteins. The regulation of mitochondrial DNA is necessary for mitochondrial biogenesis. We have selectively deleted TFAM in the megakaryocyte lineage using the Platelet factor 4 promoter driven Cre‐Lox system. This model will be very useful, and it supports our in vitro studies with metabolic inhibitors in understanding the role of mitochondria and oxidative phosphorylation in platelet function in platelet bioenergetics contributing to the thrombosisSupport or Funding InformationSupported by HL56652, HL138179 and VA Merit AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metformin Has Direct Protective Effect on Human Cardiac Mitochondria

ObjectiveMetformin (Metf), an oral antihyperglycemic agent, is widely used to treat type 2 diabetes mellitus. The primary action of the drug is to lower hepatic glucose synthesis and enhance peripheral glucose uptake via activation of the insulin receptor. It also exerts a cardioprotective effect independent of its glucose lowering action; however, mechanisms underlying cardioprotection in the human heart remain poorly defined. We hypothesize that the cardioprotective effect of Metf is associated with mild inhibition of mitochondrial oxidative phosphorylation (OXPHOS) and reduction of oxidative stress that protect mitochondria against permeability transition pore (PTP) opening.MethodsLeft atrial appendage tissue was collected from middle‐aged and elderly patients (n=21) undergoing open‐heart surgery. Mitochondria were isolated from fresh tissue using differential centrifugation and exposed to different concentrations of Metf (0–10 mM). The functional activity of mitochondrial OXPHOS complexes (CI‐V) was measured spectrophotometrically using specific OXPHOS inhibitors, electron donors, and acceptors. Superoxide production was measured by monitoring changes in fluorescence intensity of dihydroethidium (DHE) in the absence and presence of antimycin A (AA, 5 μM). The sensitivity of mitochondria toward PTP opening was assessed by exposing isolated mitochondria to 10 μM of Ca2+ pulses and monitoring abrupt mitochondrial Ca2+ release (Fluo‐5N fluorescence) and swelling of the matrix (decrease in absorbance). The study was approved by the Aurora Institutional Review Board and adhered to the Health Insurance Portability and Accountability Act and Aurora Health Care patient privacy and security guidelines. The study followed the principles of the Declaration of Helsinki.ResultsMetf had a dose‐dependent mild inhibitory effect on the activity of CI, CIV, and CV without affecting CII and CIII (Figure A). This was associated with reduction of superoxide production before and after exposure of the mitochondria to AA (Figure B). Metf in a dose‐dependent manner also attenuated Ca2+‐induced mPTP opening (Figure C) with delayed swelling of mitochondrial matrix (Figure D).ConclusionMetf improves cardioprotection by increasing tolerance to stress‐induced PTP opening due to its mild inhibitory effect on the OXPHOS and reduction of superoxide production. This can be a beneficial alternative to patients with impaired endogenous cardioprotective responses.Support or Funding InformationN 570‐3657, Cardiac Research Award, Aurora Health CareThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prolonged Cold-Ischemia Increases Reactive Oxygen Species and Activates Autophagy by Enhancing Glycolysis in the Graft through the mTOR Signaling Pathway in Orthotopic Lung Transplantation of Rats

Purpose Ischemia-reperfusion (I/R) injury continues to plague the field of graft transplantation and is a major contributor to early graft dysfunction or failure after organ transplantation. The aim of the present study is to investigate the mechanisms by which prolonged CI influences the autophagy in the graft lung, in which the mTOR signaling pathway was involved. Methods Orthotopic lung transplantation models were induced in rats, whose lungs were preserved under CI conditions for varying durations. Autophagy activities, energy metabolism, and reactive oxygen species (ROS) level were determined at each time point. The perfusion treatments of inhibitor (3-Methyladenine [3-MA]) and activator (rapamycin) of autophagy and inhibitor of oxidative phosphorylation (oligomycin [OM]) were performed to verify the relationships among mTOR signaling pathway and autophagy in the cold I/R injury. Results Autophagy was induced by CI preservation, which was peaked at 12 h after CI. Additionally, a coexistence of reduced oxidative phosphorylation and elevated ROS level was observed in rat lung tissues. Increased activities of key glycolytic enzymes, glucose consumption and lactic acid production followed with prolonged CI, which became stable after a 9 h period of CI. Of importance, the perfusion treatment of 3-MA or OM decreased ROS level and Beclin-1, PINK1, Parkin, and AMPK expression, but increased mTOR expression; while the perfusion treatment of rapamycin induced a reverse tendency. Conclusion These findings demonstrate that activated autophagy during prolonged CI preservation leads to poor oxidative phosphorylation, robust glycolysis, and ROS level rise in the graft lung via the mTOR signaling pathway.

Design of a multi-sensor platform for integrating extracellular acidification rate with multi-metabolite flux measurement for small biological samples

Rates of cellular oxygen consumption (OCR) and extracellular acidification (ECAR) are widely used proxies for mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic rate in cell metabolism studies. However, ECAR can result from both oxidative metabolism (carbonic acid formation) and glycolysis (lactate release), potentially leading to erroneous conclusions about metabolic substrate utilization. Co-measurement of extracellular glucose and lactate flux along with OCR and ECAR can improve the accuracy and provide better insight into cellular metabolic processes but is currently not feasible with any commercially available instrumentation. Herein, we present a miniaturized multi-sensor platform capable of real-time monitoring of OCR and ECAR along with extracellular lactate and glucose flux for small biological samples such as single equine embryos. This multiplexed approach enables validation of ECAR resulting from OXPHOS versus glycolysis, and expression of metabolic flux ratios that provide further insight into cellular substrate utilization. We demonstrate expected shifts in embryo metabolism during development and in response to OXPHOS inhibition as a model system for monitoring metabolic plasticity in very small biological samples. Furthermore, we also present a preliminary interference analysis of the multi-sensor platform to allow better understanding of sensor interference in the proposed multi-sensor platform. The capability of the platform is illustrated with measurements of multi-metabolites of single-cell equine embryos for assisted reproduction technologies. However, this platform has a wide potential utility for analyzing small biological samples such as single cells and tumor biopsies for immunology and cancer research applications.

YIA19-004: Collateral Deletion of Glycolysis Genes Generates Selective Vulnerabilities to Inhibitors of Oxidative Phosphorylation

Targeted therapies attacking specific genetic alterations in tumor cells have been an effective strategy in the treatment of many cancers and underlie the concept of “precision medicine.” Successes in various cancers have resulted almost exclusively from targeting activated oncogenes, but this approach has not been proven effective in patients with glioblastoma. We have taken a radically different approach to molecular therapy by targeting genomic deletions, which heretofore have not been considered to be therapeutically actionable. In a chemical biology drug screen of gliomas with passenger deletions in the glycolytic gene Enolase 1 (ENO1), we found that the most cytotoxic agents were those that inhibited mitochondrial oxidative phosphorylation (OxPhos), including a new drug developed at MD Anderson Cancer Center by the Institute of Applied Cancer Science (IACS), which inhibits OxPhos by binding mitochondrial complex I with nM affinity. Exquisite sensitivity to OxPhos inhibition derives from impaired glycolysis, as ENO1-deleted cells are unable to compensatory upregulate glycolysis in the face of inhibition of OxPhos, and succumb to bioenergetic failure. In addition to ENO1-deletions, we find that passenger deletion of other metabolic genes, including 6-phosphogluconate dehydrogenase (PGD), also exhibit selective sensitivity to OxPhos inhibition by the same underlying biochemical mechanism. The IACS compound was found to penetrate the blood brain-barrier and to eradicate PGD and ENO1-deleted intracranial xenografts. The lead OxPhos inhibitor, IACS-10759, is currently in phase 1 clinical trial for leukemia, and our data provide a strong rationale for advancing this agent to clinical trials in glioblastoma patients.

Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma

Cancer specific inhibitors reflective of unique metabolic needs, are rare. We describe a novel small molecule, Gboxin, that specifically inhibits primary mouse and human glioblastoma (GBM) cell growth but not mouse embryo fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises GBM oxygen consumption. Reliant on its positive charge, Gboxin associates with mitochondrial oxidative phosphorylation complexes in a proton gradient dependent manner and inhibits F0F1 ATP synthase activity. Gboxin resistant cells require a functional mitochondrial permeability transition pore that regulates pH impeding matrix accumulation. Administration of a pharmacologically stable Gboxin analog inhibits GBM allografts and patient derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin and exposes the elevated proton gradient pH in cancer cell mitochondria as a new mode of action for antitumor reagent development.

Fhit\u2013Fdxr interaction in the mitochondria: modulation of reactive oxygen species generation and apoptosis in cancer cells

Fhit protein is lost in cancers of most, perhaps all, cancer types; when restored, it can induce apoptosis and suppress tumorigenicity, as shown in vitro and in mouse tumor models in vivo. Following protein cross-linking and proteomics analyses, we characterized a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes the heat-shock chaperonin pair, HSP60/10, which is likely involved in importing Fhit into the mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin, in electron transport chain complex III. Overexpression of Fhit protein in Fhit-deficient cancer cells modulates the production of intracellular reactive oxygen species, causing increased ROS, following peroxide treatment, with subsequent increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape ROS overproduction and ROS-induced apoptosis, likely carrying oxidative damage. Thus, characterization of Fhit-interacting proteins has identified direct effectors of a Fhit-mediated apoptotic signal pathway that is lost in many cancers. This is of translational interest considering the very recent emphasis in a number of high-profile publications, concerning the role of oxidative phosphorylation in the treatment of human cancers, and especially cancer stem cells that rely upon oxidative phosphorylation for survival. Additionally, we have shown that cells from a Fhit-deficient lung cancer cell line, are sensitive to killing by exposure to atovaquone, thought to act as a selective oxidative phosphorylation inhibitor by targeting the CoQ10 dependence of the mitochondrial complex III, while the Fhit-expressing sister clone is resistant to this treatment.

Hypoxia-Modified Cancer Cell Metabolism.

While oxygen is critical to the continued existence of complex organisms, extreme levels of oxygen within a system, known as hypoxia (low levels of oxygen) and hyperoxia (excessive levels of oxygen), potentially promote stress within a defined biological environment. The consequences of tissue hypoxia, a result of a defective oxygen supply, vary in response to the gravity, extent and environment of the malfunction. Persistent pathological hypoxia is incompatible with normal biological functions, and as a result, multicellular organisms have been compelled to develop both organism-wide and cellular-level hypoxia solutions. Both direct, including oxidative phosphorylation down-regulation and inhibition of fatty-acid desaturation, and indirect processes, including altered hypoxia-sensitive transcription factor expression, facilitate the metabolic modifications that occur in response to hypoxia. Due to the dysfunctional vasculature associated with large areas of some cancers, sections of these tumors continue to develop in hypoxic environments. Crucial to drug development, a robust understanding of the significance of these metabolism changes will facilitate our understanding of cancer cell survival. This review defines our current knowledge base of several of the hypoxia-instigated modifications in cancer cell metabolism and exemplifies the correlation between metabolic change and its support of the hypoxic-adapted malignancy.

Bioenergetic Changes Underline Plasticity of Murine Embryonic Stem Cells.

Murine embryonic stem cells (mESCs) are endowed by a time-dependent window of plasticity during their early commitment steps. Indeed, while mESCs deprived of leukemia inhibitory factor (LIF) for 24 hours revert to their naive pluripotent state after subsequent LIF readdition, cells deprived of LIF for 48 hours are no longer efficient in reverting, upon LIF addition, and undergo irreversible differentiation. We investigated undisclosed bioenergetic profiles of early mESC-derived committed cells versus their undifferentiated states in order to reveal specific bioenergetic changes associated with mESC plasticity. Multiparametric bioenergetic analysis revealed that pluripotent (+LIF) and reversibly committed cells (-LIF24h) are energetically flexible, depending on both oxidative phosphorylation (OXPHOS) and glycolysis. They exhibit high mitochondrial respiration in the presence of the main energetic substrates and can also rely on glycolysis in the presence of OXPHOS inhibitor. Inhibition of the glycolysis or mitochondrial respiration does not change drastically the expression of pluripotency genes, which remain well expressed. In addition, cells treated with these inhibitors keep their capacity to differentiate efficiently upon embryoid bodies formation. Transition from metabolically active mESCs to irreversibly committed cells is associated with a clear change in mitochondrial network morphology, to an increase of adenosine triphosphate (ATP) produced from glycolysis and a decline of ATP turnover and of the mitochondrial activity without change in the mitochondrial mass. Our study pointed that plasticity window of mESCs is associated with the bivalent energetic metabolism and potency to shift to glycolysis or OXPHOS on demand. LIF removal provokes glycolytic metabolic orientation and consecutive loss of the LIF-dependent reversion of cells to the pluripotent state. Stem Cells 2019;37:463-475.

Organic anion transporters 1 and 3 influence cellular energy metabolism in renal proximal tubule cells.

Organic anion transporters (OATs) 1 and 3 are, besides being uptake transporters, key in several cellular metabolic pathways. The underlying mechanisms are largely unknown. Hence, we used human conditionally immortalized proximal tubule epithelial cells (ciPTEC) overexpressing OAT1 or OAT3 to gain insight into these mechanisms. In ciPTEC-OAT1 and -OAT3, extracellular lactate levels were decreased (by 77% and 71%, respectively), while intracellular ATP levels remained unchanged, suggesting a shift towards an oxidative phenotype upon OAT1 or OAT3 overexpression. This was confirmed by increased respiration of ciPTEC-OAT1 and -OAT3 (1.4-fold), a decreased sensitivity to respiratory inhibition, and characterized by a higher demand on mitochondrial oxidative capacity. In-depth profiling of tricarboxylic acid (TCA) cycle metabolites revealed reduced levels of intermediates converging into α-ketoglutarate in ciPTEC-OAT1 and -OAT3, which via 2-hydroxyglutarate metabolism explains the increased respiration. These interactions with TCA cycle metabolites were in agreement with metabolomic network modeling studies published earlier. Further studies using OAT or oxidative phosphorylation (OXPHOS) inhibitors confirmed our idea that OATs are responsible for increased use and synthesis of α-ketoglutarate. In conclusion, our results indicate an increased α-ketoglutarate efflux by OAT1 and OAT3, resulting in a metabolic shift towards an oxidative phenotype.

JMJD1C-mediated metabolic dysregulation contributes to HOXA9-dependent leukemogenesis.

Abnormal metabolism is a fundamental hallmark of cancer and represents a therapeutic opportunity, yet its regulation by oncogenes remains poorly understood. Here, we uncover that JMJD1C, a jumonji C (JmjC)-containing H3K9 demethylase, is a critical regulator of aberrant metabolic processes in homeobox A9 (HOXA9)-dependent acute myeloid leukemia (AML). JMJD1C overexpression increases in vivo cell proliferation and tumorigenicity through demethylase-independent upregulation of a glycolytic and oxidative program, which sustains leukemic cell bioenergetics and contributes to an aggressive AML phenotype in vivo. Targeting JMJD1C-mediated metabolism via pharmacologic inhibition of glycolysis and oxidative phosphorylation led to ATP depletion, induced necrosis/apoptosis and decreased tumor growth in vivo in leukemias co-expressing JMJD1C and HOXA9. The anti-metabolic therapy effectively diminished AML stem/progenitor cells and reduced tumor burden in a primary AML patient-derived xenograft. Our data establish a direct link between drug responses and endogenous expression of JMJD1C and HOXA9 in human AML cell line- and patient-derived xenografts. These findings demonstrate a previously unappreciated role for JMJD1C in counteracting adverse metabolic changes and retaining the metabolic integrity during tumorigenesis, which can be exploited therapeutically.