BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

The traditional view is that cancer cells predominately produce ATP by glycolysis, rather than by oxidation of energy-providing substrates. Mitochondrial uncoupling -the continuing reduction of oxygen without ATP synthesis -has recently been shown in leukemia cells to circumvent the ability of oxygen to inhibit glycolysis, and may promote the metabolic preference for glycolysis by shifting from pyruvate oxidation to fatty acid oxidation (FAO). Here we have demonstrated that pharmacologic inhibition of FAO with etomoxir or ranolazine inhibited proliferation and sensitized human leukemia cells -cultured alone or on bone marrow stromal cells -to apoptosis induction by ABT-737, a molecule that releases proapoptotic Bcl-2 proteins such as Bak from antiapoptotic family members. Likewise, treatment with the fatty acid synthase/lipolysis inhibitor orlistat also sensitized leukemia cells to ABT-737, which supports the notion that fatty acids promote cell survival. Mechanistically, we generated evidence suggesting that FAO regulates the activity of Bak-dependent mitochondrial permeability transition. Importantly, etomoxir decreased the number of quiescent leukemia progenitor cells in approximately 50% of primary human acute myeloid leukemia samples and, when combined with either ABT-737 or cytosine arabinoside, provided substantial therapeutic benefit in a murine model of leukemia. The results support the concept of FAO inhibitors as a therapeutic strategy in hematological malignancies. IntroductionMore than half a century ago, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that circumvents the Pasteur effect, i.e., the inhibition of anaerobic fermentation by oxygen (1). However, we have recently demonstrated that in leukemia cells, mitochondrial uncoupling - the continuing reduction of oxygen without the synthesis of ATP - could mimic the Warburg effect in the absence of permanent, transmissible alterations to the oxidative capacity of cells (2). This metabolic pattern was observed when leukemia cells were cultured on feeder layers of bone marrow-derived mesenchymal stromal cells (MSCs). MSCs have previously been reported to support both normal and malignant hematopoiesis (reviewed in refs. 3-5) and have become an important component in the in vitro modeling of the bone marrow microenvironment. Leukemia cells cultured on MSC feeder layers demonstrated increased lactate generation, and, most curiously, decreased mitochondrial membrane potential in the presence of a transient (6-8 hour) increase in oxygen consumption. Additionally, this uncoupled phenotype appeared to be associated with the antiapoptotic effect of MSC feeder layers, and we hypothesized a shift away from the complete oxidation of glucose. This concept has already been alluded to by Lynen (6), and by Ronzoni and Ehrenfest in experiments using the prototypical protonophore 2,4-dinitrophenol, and suggests a metabolic shift to fatty acid oxidation (FAO) rather than pyruvate oxidation (2, 7). Although i...

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IntroductionNormal and leukemic hematopoietic cells and stem cells reside in the bone marrow in specialized areas ("niches") that provide the structural and physiologic conditions for their growth and survival. 1 Subpopulations of leukemic cells can be sequestered in niches and thereby evade chemotherapy-induced death. 2 We and others have reported that stromal cells protect acute myeloid leukemia (AML) and chronic lymphocytic leukemia cells from the apoptosis induced by chemotherapy. [3][4][5][6] While the mechanisms of stroma-mediated protection are pleiotropic and involve a complex interplay of stroma-produced cytokines, chemokines, and adhesion molecules, the stroma-secreted chemokine stromal-derived factor 1␣ (SDF-1␣) and its cognate receptor CXCR4 have recently emerged as critical mediators of stromal/leukemic cell interactions. 7,8 SDF-1␣ and CXCR4 primarily regulate the migration, homing, and mobilization of hematopoietic cells. 9,10 Binding of SDF-1␣ to CXCR4 causes CXCR4 to be incorporated into lipid rafts 11 and increases its phosphorylation. 12 The latter leads to prolonged activation of the extracellular signaling-regulated kinase (ERK) and phosphoinositol 3-kinase (PI3K) pathways, 13 which are key signaling pathways that promote leukemia cells survival. 14,15 Both surface and intracellular 16 CXCR4 levels were found to be elevated in a subset of AML cases. Further, CXCR4 has been shown to mediate the homing and engraftment of AML cells to the bone marrow of nonobese diabetes (NOD)/severe combined immunodeficiency (SCID) mice. 17,18 Finally, CXCR4 was recently reported to be expressed at higher levels in cases of AML associated with an internal tandem duplication (ITD) type of mutation of the gene that encodes fetal liver tyrosine . 19 This is one of the most frequent mutations in AML, which confers poor response to chemotherapy and only transient response to FLT3 inhibitors. 20,21 Our recent studies, in addition, indicated that CXCR4 expression is associated with poor prognosis in patients with diploid AML regardless of FLT3 mutation status. 22,23 Altogether, these findings suggest that disruption of these interactions by SDF-1␣/CXCR4 antagonists represents a novel strategy for targeting leukemia/bone marrow microenvironment interactions. We have reported that inhibition of CXCR4 by specific synthetic peptides (ie, RCP168) interferes with stromal/ leukemic cell interactions and increases the sensitivity of leukemic cells to chemotherapy. 24 In this study, we used AMD3465 (Anormed and Genzyme, Cambridge, MA), a second-generation smallmolecule reversible inhibitor of SDF-1␣/CXCR4 with a half maximal inhibitory concentration (IC 50 An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use on...