Targeting Anaplerotic Pathways That Support Fatty Acid Metabolism as a Therapeutic Strategy for Hematological Malignancies: The Achilles’ Heel of the Warburg Effect.
Authors: Samudio Ismael J. , Fiegl Michael , Konopleva Marina , Kaluarachchi Kumar , McMurray John S. , Korchin Borys , Dwyer Karen Clise , Harmancey Romain , Guthrie Patrick , Taegtmeyer Heinrich , Andreeff Michael 
Published: April 2020

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.