Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition. A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis. To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells. Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. Disclosures Minden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

TAMI-13. MODULATION OF REDUCTIVE CARBOXYLATION FLUX OF GLUTAMINE IN GLIOBLASTOMA CELLS BY USING OSCILLATING MAGNETIC FIELD

Increased cell proliferation in glioblastoma (GBM) leads to hypoxia in the tumor microenvironment. This is a major concern in GBM patients as it promotes tumor invasion. Glutaminolysis is a hallmark of cancer cells and under hypoxic conditions glutamine metabolism proceeds through reductive carboxylation pathway. Recently, we have shown that oscillating magnetic field (OMF) produces oncolytic effects which can influence cellular metabolism. Here, we have explored the effect of OMF on glutamine metabolism in GBM cells. Patient-derived GBM cells were grown in high glucose (25 mM) DMEM supplemented with 20% fetal bovine serum (FBS), 2.0 mM glutamine and 1.0 mM pyruvate at 37 °C under humidified air and 5% CO2. Cells were divided into 2 groups (Test and Sham; n = 4 each group). When reached confluency (~2.0×106 cells/mL), cells in both groups were treated with 4.0 mM of [U-13C]glutamine in DMEM (supplemented with 20% FBS, and 1.0 mM pyruvate). The “Test” group was subjected to OMF for 3 hours, and the “Sham” group was treated similar to the “Test” group but with non-magnetic rods of the same dimensions as the magnets in the Test group. After 3 h, cells were harvested in 50% methanol analyzed by GC-MS. The 13C-isotopomer analysis showed that glutamine metabolism in GBM cells proceeds through reduction carboxylation, confirmed by the higher levels of M+5 citrate (15.42 ± 1.28 % ) than M+4 citrate (2.05 ± 0.28 %). When GBM cells were treated with OMF, a statistically significant decrease in the citrate M+5 was observed, compared to the “Sham” treated group (15.42 ± 1.28 % vs. 8.89 ± 1.30 %; p = 0.0003). This decrease in M+5 citrate upon OMF treatment clearly indicates that the OMF decreases the reductive carboxylation flux of glutamine in GBM cells which would have therapeutic value in treating GBM patients.

Reprogramming the neuroblastoma epigenome with a mitochondrial uncoupler

Dysregulated DNA methylation is associated with poor prognosis in cancer patients, promoting tumorigenesis and therapeutic resistance1. DNA methyltransferase inhibitors (DNMTi) reduce DNA methylation and promote cancer cell differentiation, with two DNMTi already approved for cancer treatment2. However, these drugs rely on cell division to dilute existing methylation, thus the ‘demethylation’ effects are achieved in a passive manner, limiting their application in slow-proliferating tumor cells. In this study we use a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to actively achieve global DNA demethylation. NEN treatment promotes DNA demethylation by activating electron transport chain (ETC) to produce α-ketoglutarate (α-KG), a substrate for the DNA demethylase TET. In addition, NEN inhibits reductive carboxylation, a key metabolic pathway to support growth of cancer cells with defective mitochondria or under hypoxia. Importantly, NEN treatment reduces 2-hydroxyglutarate (2-HG) generation and blocks DNA hypermethylation under hypoxia. Together, these metabolic reprogramming effects of NEN actively alter the global DNA methylation landscape and promote neuroblastoma differentiation. These results not only support Warburg’s original hypothesis that inhibition of ETC causes cell de-differentiation and tumorigenesis, but also suggest that mitochondrial uncoupling is an effective metabolic and epigenetic intervention that remodels the tumor epigenome for better prognosis.

Cyanide as a Primordial Reductant enables a Protometabolic Reductive Glyoxylate Pathway

Investigation of prebiotic chemical pathways leading to protometabolic forerunners of metabolism has been largely based on bio-inspired (iron-mediated) reductive conversion of carbon dioxide and of carboxylic acid substrates.1,2 While attractive from a parsimony point of view, this approach has been challenging with debatable outcomes.3,4 Herein, we show that cyanide reacts with citric acid cycle (TCA) intermediates and derivatives and acts as a primordial reducing agent mediating abiotic reductive transformations. The hydrolysis of the cyanide adducts followed by decarboxylation enables the efficient reductive-decarboxylative transformation of oxaloacetate to malate and fumarate to succinate while pyruvate and α-ketoglutarate are not reduced. In the presence of glyoxylate,5,6 malonate7 and malononitrile,8 alternative pathways emerge, which after decarboxylation produce metabolic intermediates and related compounds also found in meteorites.9 These results, along with the previous demonstration of the metal-free alpha-keto analog of the reverse-TCA cycle,4,6 suggest that (a) alternative paradigms of cyanide-based protometabolic reactions bypassing the abiotic reductive-carboxylation steps can be prebiotically viable, (b) a novel reductive glyoxylate pathway can be a precursor to the r-TCA cycle and (c) the type of sophisticated carboxylation and reduction chemistries which are part of extant metabolic cycles10,11 are an evolutionary invention mediated by complex metalloproteins11.

Hypoxia uncouples HIF gene transcription and metabolic flux in proliferating primary cells

Hypoxia is an important environmental stimulus that causes transcriptional and metabolic reprogramming in cells to facilitate their survival. Here, we performed stable isotope tracing and metabolic flux analyses of proliferating primary cells in hypoxia. Despite activation of the hypoxia-inducible factor (HIF) transcriptional program and up-regulation of glycolytic genes, glycolytic flux was decreased in hypoxic cells in our models. No evidence for increased glutaminolysis or reductive carboxylation was observed. While pharmacologic stabilization of HIF in normoxia with the prolyl hydroxylase inhibitor molidustat did increase glycolytic flux as expected, hypoxia abrogated this effect. Together, these data suggest that primary cell bioenergetic metabolism is closely coupled to cell proliferation rate and that other regulatory factors override the effects of HIF-dependent up-regulation of glycolytic gene expression on glycolytic flux.

Abstract 308: Reductive Carboxylation Contributes to Cardiac Adaptation in Response to the Oncometabolite D2-hydroxyglutarate

Cancer cells rewire metabolism to support tumor growth and proliferation. In isocitrate dehydrogenase (IDH) 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. D2-HG causes inhibition of alpha-ketoglutarate dehydrogenase (AKGDH), which is associated with reduced cardiac contractile function. How tumor cells influence the metabolism of cardiomyocytes remains mostly unknown. Specific cancer cells use glutamine-dependent reductive carboxylation to circumvent defective mitochondrial metabolism by producing citrate and acetyl-CoA for lipid synthesis, which tumors require for growth. Here, we explore the hypothesis that inhibition of AKGDH by the oncometabolite D2-HG increases glutamine-dependent reductive carboxylation in the heart. We combined ex vivo rat heart perfusions with mass-spectrometry-based stable isotope tracer studies and in silico metabolic flux analysis. In response to D2-HG-mediated inhibition of AKGDH, we observed an increased reductive carboxylation of alpha-ketoglutarate to citrate rather than oxidative decarboxylation. This pathway increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. When we perfused rat hearts with 13C-labelled D2-HG, we observed a similarly increased formation of citrate. To identify which IDH isoform is responsible for redirecting carbon flux, we modulated IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes using siRNAs. Reduced expression of IDH1 impaired reductive formation of citrate. Importantly, we observed a significant correlation between reductive citrate formation and epigenetic modifications of histones, including increased histone 3 lysine 9 acetylation and di-methylation. To explore these observations, we conducted ChIP-sequencing and identified distinct transcriptional remodeling. Taken together, we demonstrate how oncometabolic stress in the heart causes redirection of central carbon metabolism via reductive carboxylation, and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.

Cancer cells with defective oxidative phosphorylation require endoplasmic reticulum–to–mitochondria Ca2+ transfer for survival

Spontaneous Ca2+ signaling from the InsP3R intracellular Ca2+ release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca2+ uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP3R activity or mitochondrial Ca2+ uptake increased α-ketoglutarate (αKG) abundance and the NAD+/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)–to–mitochondria Ca2+ transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca2+ inhibited αKGDH activity and increased NAD+, which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca2+ from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca2+ regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca2+ transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.