Pharmacological Inhibition of Fatty Acid Oxidation as a Novel Therapeutic Concept for Acute Myeloid Leukemia.

Abstract Abstract 3779 Poster Board III-715 Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect, i.e. the inhibition of anaerobic fermentation by oxygen. We have recently demonstrated in leukemia cells that mitochondrial uncoupling, i.e. the abrogation of ATP synthesis in response to mitochondrial membrane potential (MMP), promotes the Warburg effect, contributes to chemo-resistance and represents a metabolic shift to fatty acid oxidation (FAO). Exposure of leukemic cells to marrow-derived mesenchymal stromal cells (MSC) promotes accumulation of lactate and reduces MMP. Stroma/leukemia co-cultures protect leukemia cells from chemotherapy-induced apoptosis. We found that he decrease in MMP was mediated by mitochondrial uncoupling accompanied by increased expression of mitochondrial uncoupling protein (UCP2) (Cancer Res. 68:5198,2008). We therefore proposed that the Warburg effect may be the result of preferential oxidation of fatty acids in cancer cell mitochondria (Cancer Res.69:2163,2009). Here we demonstrate that leukemia cells uncouple FAO from ATP synthesis, and that pharmacological inhibition of FAO with etomoxir or ranolazine inhibits proliferation and sensitizes leukemia cells – cultured alone or on bone marrow stromal cells – to apoptosis induction by the BH3 mimetic ABT-737 and the MDM-2 antagonist Nutlin 3a. Results suggest that leukemia cells rely, at least in part, on de novo fatty acid synthesis (FAS) to support FAO. Furthermore, treatment with the FAS inhibitor orlistat sensitized leukemia cells to apoptosis induction by ABT-737. Mechanistically, mitochondria derived from etomoxir treated leukemia cells were sensitized to release of cytochrome C and apoptosis-inducing-factor (AIF) upon treatment with ABT-737. Etomoxir (EX) facilitated the formation of Bak oligomers after treatment with ABT-737 suggesting that FAO regulates the activity of Bak-dependent mitochondrial permeability transition. Lastly, we present evidence that EX, in combination with liposomal ABT-737 or cytosine arabinoside (AraC), provides significant therapeutic benefit in a murine model of human leukemia (luciferase/GFP marked MOLT13 cells) as evidenced by reduced in vivo growth kinetics (BLI) and prolonged median survival (ABT-737 vs. EX+ABT-737, p=<0.05; AraC vs.EX+AraC,p<0.0001 ). Conclusions: 1) results support the notion that the Warburg effect may be the result of preferential oxidation of fatty acids by leukemia mitochondria. 2) Inhibition of fatty acid oxidation is proposed as a novel therapeutic concept for hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

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Targeting Anaplerotic Pathways That Support Fatty Acid Metabolism as a Therapeutic Strategy for Hematological Malignancies: The Achilles’ Heel of the Warburg Effect.

Blood ◽

10.1182/blood.v112.11.1631.1631 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1631-1631

Author(s):

Ismael J. Samudio ◽

Michael Fiegl ◽

Marina Konopleva ◽

Kumar Kaluarachchi ◽

John S. McMurray ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Cancer Cells ◽

Fatty Acid Oxidation ◽

Therapeutic Strategy ◽

Leukemia Cell ◽

Atp Synthesis ◽

Leukemia Cells ◽

Acid Oxidation ◽

Mitochondrial Uncoupling

Abstract More than half a century ago, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect, i.e. the inhibition of anaerobic fermentation by oxygen. However, permanent and transmissible defects in the respiratory capacity of cancer cells that could broadly support Warburg’s hypothesis have not been identified. Notably, we have recently demonstrated that mitochondrial uncoupling – the abrogation of ATP synthesis in response to mitochondrial membrane potential – can promote the Warburg effect in leukemia cells, and may contribute to chemoresistance, via in part, the expression of the highly conserved thermogenic protein UCP2. Here we demonstrate that mitochondrial uncoupling in leukemia cells is supported by the oxidation of fatty acids, and provide evidence that etomoxir (EX) or ranolazine (RAN), pharmacological inhibitors of fatty acid oxidation utilized for the treatment of heart failure, sensitize leukemia cell lines and primary samples to apoptosis induced by the BH3 mimetic ABT-737 and the MDM-2 inhibitor Nutlin 3a. EX and RAN, but not 2-deoxyglucose (2DG), markedly inhibited oxygen consumption in leukemia cell lines and primary samples. In contrast, 2DG, but not EX or RAN, potently depleted ATP levels, suggesting that the oxidation of fatty acids is uncoupled from ATP synthesis – and conversely, the synthesis of ATP primarily depends on the non-oxidative, glycolytic metabolism of glucose. It is noteworthy that albeit EX and RAN inhibited the growth of p53-wild type and -mutant leukemia cells, neither agent induced marked apoptosis. Nonetheless, a pronounced induction of the proapoptotic BH3-only proteins Noxa and Bim was observed regardless of p53 status, suggesting a potential mechanism by which these agents enhance apoptosis by ABT-737. In addition, EX and RAN abrogated the chemoprotective effects of bone marrow-derived stromal feeder layers, and EX provided a survival advantage in combination with ABT-737 in a murine model of leukemia suggesting that inhibition of mitochondrial fatty acid oxidation represents a novel therapeutic strategy for the treatment of leukemia. Intriguingly, C13-NMR analysis, H3–oleate oxidation, and oxymetry experiments revealed that leukemia cells do not oxidize exogenous fatty acids, but rather depend on glucose and glutamine-supported de novo synthesis of fatty acids to maintain mitochondrial function. Accordingly, depletion of glutamine, inhibition of fatty acid synthesis, or reduced pentose phosphate shunt-derived NADPH significantly decreased oxygen consumption and potentiated ABT-737 induced apoptosis. The above results support the hypothesis that glutamine and glucose-dependent anaplerotic reactions sustain fatty acid metabolism and survival of leukemia cells. Our results suggest that the dependence of cancer cells on glycolysis for energy generation indicates a metabolic shift to the ATP-uncoupled oxidation of non-glucose substrates, and most importantly, support the clinical investigation of fatty acid oxidation and synthesis inhibitors as a therapeutic strategy in hematological malignancies.

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3,5-Diiodo-l-thyronine rapidly enhances mitochondrial fatty acid oxidation rate and thermogenesis in rat skeletal muscle: AMP-activated protein kinase involvement

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90642.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. E497-E502 ◽

Cited By ~ 57

Author(s):

A. Lombardi ◽

P. de Lange ◽

E. Silvestri ◽

R. A. Busiello ◽

A. Lanni ◽

...

Keyword(s):

Skeletal Muscle ◽

Fatty Acids ◽

Fatty Acid ◽

Energy Metabolism ◽

Fatty Acid Oxidation ◽

Atp Synthesis ◽

Acid Oxidation ◽

Mitochondrial Fatty Acid Oxidation ◽

Mitochondrial Fatty Acid ◽

Amp Activated Protein Kinase

Triiodothyronine regulates energy metabolism and thermogenesis. Among triiodothyronine derivatives, 3,5-diiodo-l-thyronine (T2) has been shown to exert marked effects on energy metabolism by acting mainly at the mitochondrial level. Here we investigated the capacity of T2 to affect both skeletal muscle mitochondrial substrate oxidation and thermogenesis within 1 h after its injection into hypothyroid rats. Administration of T2 induced an increase in mitochondrial oxidation when palmitoyl-CoA (+104%), palmitoylcarnitine (+80%), or succinate (+30%) was used as substrate, but it had no effect when pyruvate was used. T2 was able to 1) activate the AMPK-ACC-malonyl-CoA metabolic signaling pathway known to direct lipid partitioning toward oxidation and 2) increase the importing of fatty acids into the mitochondrion. These results suggest that T2 stimulates mitochondrial fatty acid oxidation by activating several metabolic pathways, such as the fatty acid import/β-oxidation cycle/FADH2-linked respiratory pathways, where fatty acids are imported. T2 also enhanced skeletal muscle mitochondrial thermogenesis by activating pathways involved in the dissipation of the proton-motive force not associated with ATP synthesis (“proton leak”), the effect being dependent on the presence of free fatty acids inside mitochondria. We conclude that skeletal muscle is a target for T2, and we propose that, by activating processes able to enhance mitochondrial fatty acid oxidation and thermogenesis, T2 could play a role in protecting skeletal muscle against excessive intramyocellular lipid storage, possibly allowing it to avoid functional disorders.

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Inhibition of Fatty Acid Oxidation with Avocatin B Selectively Targets AML Cells and Leukemia Stem Cells

Blood ◽

10.1182/blood.v124.21.268.268 ◽

2014 ◽

Vol 124 (21) ◽

pp. 268-268 ◽

Cited By ~ 4

Author(s):

Eric A Lee ◽

Leonard Angka ◽

Sarah-Grace Rota ◽

Thomas Hanlon ◽

Rose Hurren ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Fatty Acid ◽

Fatty Acid Oxidation ◽

Leukemia Cell ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Acid Oxidation ◽

Selective Toxicity ◽

Induced Apoptosis

Abstract Acute myeloid leukemia (AML) is an aggressive malignant disease characterized by poor patient outcome and suboptimal front-line chemotherapy. To identify novel anti-AML compounds, we performed a high-throughput screen of a natural products library (n=800). This screen was performed against the AML cell line (TEX), which has several properties of leukemia stem cells, the cells responsible for disease pathophysiology and patient relapse. Here, avocatin B was identified as a potent and novel anti-leukemia agent. Avocatin B, at concentrations as high as 20µM, had no effect on normal peripheral blood stem cell viability. In contrast, it induced death of primary AML cells with an EC50 of 1.5-5.0 µM. Selective toxicity towards a functionally defined subset of primitive leukemia cells was also demonstrated. Avocatin B (3µM) reduced clonogenic growth of AML progenitor cells with no effect on clonogenic growth of normal hematopoietic stem cells. Further, treatment of primary AML cells with avocatin B (3µM) diminished their ability to engraft into the bone marrow of pre-conditioned, NOD/SCID mice (t18=6.5; p<0.001). Together, these results confirm that avocatin B is a novel anti-AML agent with selective toxicity toward leukemia and leukemia stem cells. Mechanistically, avocatin B-induced reactive oxygen species (ROS)-dependent leukemia cell apoptosis that was characterized by the release of mitochondrial proteins, cytochrome c and apoptosis inducing factor (AIF). Cytochrome c and AIF were detected in the cytosol of avocatin B treated TEX cells by flow cytometry. Avocatin B-induced apoptosis, as measured by the Annexin V/Propidium iodide assay, DNA fragmentation and PARP cleavage, was abolished in the presence of anti-oxidants confirming the functional importance of ROS. Next, we further evaluated the role of mitochondria in avocatin B-induced apoptosis. First, we generated leukemia cells lacking mitochondria by successive culturing in media containing ethidium bromide. The drastic (>80%) reduction in mitochondria were confirmed by nonyl acridine orange staining and flow cytometry and a near absence of the mitochondria specific proteins ANT and ND1, as measured by Western blotting. Avocatin B’s activity was abolished in leukemia cells lacking mitochondria. Next, using lentiviral knockdown, we generated leukemia cells lacking CPT1, the enzyme that facilitates transport of 16-20 carbon lipids into mitochondria. Avocatin B’s activity was abolished in cells with reduced CPT1 expression (>70% as measured by qPCR analysis). To further confirm the importance of CPT1 in avocatin B-induced death, we chemically inhibited CPT1 with etomoxir. Avocatin B’s activity was blocked in the presence of etomoxir, further demonstrating that avocatin B accumulates in mitochondria. Since avocatin B is a lipid that targets mitochondria and that mitochondria can oxidize fatty acids for energy, we next assessed the impact of avocatin B on fatty acid oxidation, using the Seahorse Bioanalyzer. Avocatin B inhibited leukemia cell fatty acid oxidation (>40% reduction in oxygen consumption at 10µM) and this occurred at a 10-fold less concentration than etomoxir, the standard experimental molecule used to probe this pathway. Further, avocatin B resulted in a 50% reduction in levels of NADPH, an important co-factor generated during fatty acid oxidation that participates in catabolic processes during cell proliferation. These results show that avocatin B accumulates in mitochondria to inhibit fatty acid oxidation and reduce NADPH to result in ROS-mediated leukemia cell apoptosis. This highlights a novel AML-therapeutic strategy by which mitochondria are targeted to impair cellular metabolism leading directly to AML cell death. Disclosures No relevant conflicts of interest to declare.

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Abstract 15465: Upregulation of Mitochondrial Atpase Inhibitory Factor 1 Mediates Increased Glycolysis in Pathological Cardiac Hypertrophy

Circulation ◽

10.1161/circ.142.suppl_3.15465 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Bo Zhou ◽

Arianne Caudal ◽

Xiaoting Tang ◽

Juan D Chavez ◽

Andrew Keller ◽

...

Keyword(s):

Fatty Acid ◽

Glucose Metabolism ◽

Cardiac Hypertrophy ◽

Fatty Acid Oxidation ◽

Atp Synthase ◽

Atp Synthesis ◽

Inhibitory Factor ◽

Acid Oxidation ◽

Mitochondrial Atpase ◽

Inhibitory Factor 1

Background: During the development of heart failure cardiac fuel metabolism switches from predominantly fatty acid oxidation (FAO) to increased reliance on glucose, especially glycolysis. Mechanisms responsible for the switch are poorly understood but appear to be coupled with impaired mitochondrial function. We recently demonstrated that increased glucose metabolism is required for cardiomyocytes growth during pathological remodeling. Hypothesis: Upregulation of mitochondrial ATPase inhibitory factor 1 (ATPIF1) in hypertrophied hearts suppresses ATP synthesis and shifts cardiac metabolism from fatty acid oxidation towards glucose metabolism. Methods and Results: We report that ATPIF1 expression is upregulated in cardiomyocytes and mouse hearts undergoing pathological hypertrophy. Using genetic models of ATPIF1 gain- and loss-of-function in cardiomyocytes and in mouse hearts,we find that upregulation of ATPIF1 in cardiac hypertrophy inhibits ATP synthesis. Furthermore, quantitative analysis of chemical crosslinking by mass spectrometry revealed that increased expression of ATPIF1 promoted the formation of F o F 1 -ATP synthase nonproductive tetramer. Impairment of F o F 1 -ATP synthase function in respiring mitochondria increasedROS generation resulting in transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the switch to glycolysis in pressure overload induced cardiac hypertrophy. Conclusions: We show that upregulation of ATPIF1 drives glucose metabolism at the expense of energy supply during the pathological growth of cardiomyocytes. Our study proposes a central role of ATP synthase in toggling anabolic and catabolic metabolism during pathological remodeling, illustrating a new concept for metabolic reprogramming of the heart.

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STUDIES OF FATTY ACID OXIDATION: 5. THE EFFECT OF DECANOIC ACID ON OXIDATIVE PHOSPHORYLATION

Canadian Journal of Biochemistry and Physiology ◽

10.1139/y56-125 ◽

1956 ◽

Vol 34 (1) ◽

pp. 1227-1232 ◽

Cited By ~ 7

Author(s):

P. G. Scholefield

Keyword(s):

Fatty Acid ◽

Oxidative Phosphorylation ◽

Rat Brain ◽

Fatty Acid Oxidation ◽

Rat Kidney ◽

Atp Synthesis ◽

Decanoic Acid ◽

Brain Mitochondria ◽

Acid Oxidation ◽

Rat Brain Mitochondria

The effects of potassium decanoate on the phosphorylation associated with the oxidation of pyruvate by rat-kidney and rat-brain mitochondria have been investigated. The suggestion that these two processes may be uncoupled from each other in the presence of decanoate has been confirmed. Further, it has been shown that the decanoate-insensitive oxidation of pyruvate by rat-brain mitochondria, occurring in the absence of such stimulating agents as fumarate, is not associated with ATP synthesis. The fumarate-stimulated oxidation of pyruvate by rat-brain mitochondria, which is inhibited by decanoate, is associated with a phosphorylation process which is uncoupled by decanoate. When pyruvate oxidation by rat-kidney or by rat-brain mitochondria is uncoupled from phosphorylation, the extent of uncoupling is proportional to the amount of decanoate added.

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Pharmacologic inhibition of fatty acid oxidation sensitizes human leukemia cells to apoptosis induction

Journal of Clinical Investigation ◽

10.1172/jci38942 ◽

2010 ◽

Vol 120 (1) ◽

pp. 142-156 ◽

Cited By ~ 371

Author(s):

Ismael Samudio ◽

Romain Harmancey ◽

Michael Fiegl ◽

Hagop Kantarjian ◽

Marina Konopleva ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Oxidation ◽

Leukemia Cells ◽

Acid Oxidation ◽

Human Leukemia ◽

Apoptosis Induction ◽

Human Leukemia Cells ◽

Pharmacologic Inhibition

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Genetic Ablation of Calcium-independent Phospholipase A2γ Prevents Obesity and Insulin Resistance during High Fat Feeding by Mitochondrial Uncoupling and Increased Adipocyte Fatty Acid Oxidation

Journal of Biological Chemistry ◽

10.1074/jbc.m110.115766 ◽

2010 ◽

Vol 285 (47) ◽

pp. 36495-36510 ◽

Cited By ~ 50

Author(s):

David J. Mancuso ◽

Harold F. Sims ◽

Kui Yang ◽

Michael A. Kiebish ◽

Xiong Su ◽

...

Keyword(s):

Insulin Resistance ◽

Fatty Acid ◽

Fatty Acid Oxidation ◽

Acid Oxidation ◽

High Fat ◽

Mitochondrial Uncoupling ◽

Genetic Ablation ◽

Fat Feeding

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Alteration of Energy Substrates and ROS Production in Diabetic Cardiomyopathy

Mediators of Inflammation ◽

10.1155/2013/461967 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 37

Author(s):

O. Lorenzo ◽

E. Ramírez ◽

B. Picatoste ◽

J. Egido ◽

J. Tuñón

Keyword(s):

Fatty Acid ◽

Fatty Acid Oxidation ◽

Diabetic Cardiomyopathy ◽

Receptor Interaction ◽

Acid Oxidation ◽

Mitochondrial Uncoupling ◽

Energy Substrates ◽

Atp Production ◽

Fatty Acid Derivatives ◽

Or Gene

Diabetic cardiomyopathy is initiated by alterations in energy substrates. Despite excess of plasma glucose and lipids, the diabetic heart almost exclusively depends on fatty acid degradation. Glycolytic enzymes and transporters are impaired by fatty acid metabolism, leading to accumulation of glucose derivatives. However, fatty acid oxidation yields lower ATP production per mole of oxygen than glucose, causing mitochondrial uncoupling and decreased energy efficiency. In addition, the oxidation of fatty acids can saturate and cause their deposition in the cytosol, where they deviate to induce toxic metabolites or gene expression by nuclear-receptor interaction. Hyperglycemia, the fatty acid oxidation pathway, and the cytosolic storage of fatty acid and glucose/fatty acid derivatives are major inducers of reactive oxygen species. However, the presence of these species can be essential for physiological responses in the diabetic myocardium.

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The transcriptional coactivator PGC-1α is essential for maximal and efficient cardiac mitochondrial fatty acid oxidation and lipid homeostasis

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00081.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. H185-H196 ◽

Cited By ~ 117

Author(s):

John J. Lehman ◽

Sihem Boudina ◽

Natasha Hausler Banke ◽

Nandakumar Sambandam ◽

Xianlin Han ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Oxidation ◽

Atp Synthesis ◽

Lipid Homeostasis ◽

Acid Oxidation ◽

Metabolic Efficiency ◽

Transcriptional Coactivator ◽

Mitochondrial Fatty Acid Oxidation ◽

Mitochondrial Fatty Acid ◽

Myocardial Fibers

High-capacity mitochondrial ATP production is essential for normal function of the adult heart, and evidence is emerging that mitochondrial derangements occur in common myocardial diseases. Previous overexpression studies have shown that the inducible transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is capable of activating postnatal cardiac myocyte mitochondrial biogenesis. Recently, we generated mice deficient in PGC-1α (PGC-1α−/− mice), which survive with modestly blunted postnatal cardiac growth. To determine if PGC-1α is essential for normal cardiac energy metabolic capacity, mitochondrial function experiments were performed on saponin-permeabilized myocardial fibers from PGC-1α−/− mice. These experiments demonstrated reduced maximal (state 3) palmitoyl-l-carnitine respiration and increased maximal (state 3) pyruvate respiration in PGC-1α−/− mice compared with PGC-1α+/+ controls. ATP synthesis rates obtained during maximal (state 3) respiration in permeabilized myocardial fibers were reduced for PGC-1α−/− mice, whereas ATP produced per oxygen consumed (ATP/O), a measure of metabolic efficiency, was decreased by 58% for PGC-1α−/− fibers. Ex vivo isolated working heart experiments demonstrated that PGC-1α−/− mice exhibited lower cardiac power, reduced palmitate oxidation, and increased reliance on glucose oxidation, with the latter likely a compensatory response. 13C NMR revealed that hearts from PGC-1α−/− mice exhibited a limited capacity to recruit triglyceride as a source for lipid oxidation during β-adrenergic challenge. Consistent with reduced mitochondrial fatty acid oxidative enzyme gene expression, the total triglyceride content was greater in hearts of PGC-1α−/− mice relative to PGC-1α+/+ following a fast. Overall, these results demonstrate that PGC-1α is essential for the maintenance of maximal, efficient cardiac mitochondrial fatty acid oxidation, ATP synthesis, and myocardial lipid homeostasis.

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