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...
• High CRM1 expression was associated with short survival of AML patients.• CRM1 inhibitor induces apoptosis mainly in a p53-dependent manner, whereas inhibition of proliferation was p53 independent.Chromosomal region maintenance 1 (CRM1) is a nuclear export receptor recognizing proteins bearing a leucine-rich nuclear export signal. CRM1 is involved in nuclear export of tumor suppressors such as p53. We investigated the prognostic significance of CRM1 in acute myeloid leukemia (AML) and effects of a novel small-molecule selective inhibitor of CRM1. CRM1 protein expression was determined in 511 newly diagnosed AML patients and was correlated with mouse double minute 2 (MDM2) and p53 levels. High CRM1 expression was associated with short survival of patients and remained an adverse prognostic factor in multivariate analysis. CRM1 inhibitor KPT-185 induced mainly full-length p53 and apoptosis in a p53-dependent manner, whereas inhibition of proliferation was p53 independent. Patient samples with p53 mutations showed low sensitivity to KPT-185. Nuclear retention of p53 induced by CRM1 inhibition synergized with increased levels of p53 induced by MDM2 inhibition in apoptosis induction. KPT-185 and Nutlin-3a, alone and in combination, induced synergistic apoptosis in patient-derived CD34 1 /CD38 -AML, but not in normal progenitor cells. Data suggest that CRM1 exerts an antiapoptotic function and is highly prognostic in AML. We propose a novel combinatorial approach for the therapy of AML, aimed at maximal activation of p53-mediated apoptosis by concomitant MDM2 and CRM1 inhibition. (Blood. 2013;121(20):4166-4174)
870 p53 is a transcription factor that prevents abnormal cell growth. Cellular levels of p53 are critically regulated by MDM2, which is frequently over-expressed in AML. Nutlin-3a disrupts MDM2-p53 interaction, increases cellular levels of p53 in both nucleus and cytoplasm, and activates p53 signaling in cells. p53 status is the major determinant of response to MDM2 inhibitors. p53 is shuttled between the nucleus and the cytoplasm, and CRM1 mediates its nuclear export. Karyopharm Therapeutics has developed novel, potent and irreversible small molecule selective inhibitors of CRM1. We hypothesized that CRM1 inhibition would enhance the nuclear activity of p53, thereby enhancing p53-mediated transcription-dependent apoptotic signaling in AML. We measured CRM1 expression in primary AML samples and investigated if blockade of nuclear export of p53 by CRM1 inhibition would enhance MDM2 inhibitor-induced apoptosis in AML. CRM1 expression was investigated in 511 patient AML samples using a validated robust reverse-phase protein array. Higher levels of CRM1 were associated with higher marrow and peripheral blast percentages (P < 0.00001). Expression was lower in those with favorable cytogenetics compared to those with intermediate or unfavorable cytogenetics (P = 0.029). CRM1 levels were higher in patients with FLT3 mutations (P = 0.003). In 3-way correlation (using distance weighted least squares), there was a clear interaction with p53 levels being highest when CRM1 was high and MDM2 levels were low. Overall survival progressively worsened as CRM1 levels increased, with median survival of 66 weeks for those with CRM1 expression in the lowest third, 47 weeks for middle third and 37 weeks in the highest third (P = 0.007). CRM1 levels did not affect remission duration (P = 0.33). The CRM1 inhibitor KPT-185 exhibited dose-dependent anti-proliferative and cytotoxic activity in AML cell lines, as evidenced by low IC50 values and high Annexin V positivity (= low ED50 values). IC50 values for wild-type p53 cells ranged from 27 to 38 nM, and for mutant p53 cells from 48 to 112 nM, suggesting that KPT-185 potently inhibits AML cell growth largely independent of p53. In contrast, apoptosis induction by KPT-185 was much more prominent in p53 wild-type than in p53-defective cells: ED50 values for Annexin V induction were 150, 90 and 85 nM in p53 wild-type and > 1000 nM in 5 of 6 p53 mutant cell lines. Stable p53 knockdown (> 90% efficiency) rendered AML cells resistant to KPT-induced apoptosis. KPT-185 induced p53 target genes TP53I3, GDF15, MDM2 and ZMAT3 partially in a p53-dependent manner. Hence, p53 was identified as major determinant of CRM1 inhibition-induced apoptosis in AML. MDM2-inhibitor Nutlin-3a induced p53 in both nucleus and cytoplasm, while CRM1 inhibition accumulated p53 in the nucleus. Treatment with KPT-185 or Nutlin-3a caused time-dependent increase in cellular p53 levels. The KPT-185/Nutlin-3a combination induced p53 more efficiently than the individual agents by accumulating p53 exclusively in the nucleus, and synergistically induced apoptosis and cell death. p53 knockdown abrogated these synergistic effects. In primary AML cells, both KPT-185 (24.7 – 36.7% Annexin V) and Nutlin-3a (13.6 – 59.8%) induced apoptosis in a dose-dependent manner. Importantly, both KPT-185 and Nutlin-3a induced apoptosis in CD34+CD38- progenitor cell populations as effectively as they did in bulk AML cells, suggesting high sensitivity of CD34+CD38- cells to CRM1 inhibition and MDM2 inhibition. KPT-185 and Nutlin-3a synergized in the induction of apoptosis in both bulk and CD34+CD38- AML progenitor cells: combination index (CI) values were 0.26 (bulk) and 0.30 (CD34+CD38-) for ED50 and 0.93 (bulk) and 0.46 (CD34+CD38-) for ED75, indicating highly synergistic (CI < 1) efficacy in apoptosis induction. The relation between p53 status and sensitivity to Nutlin-induced apoptosis has been well established. Nutlin-resistant samples were much less sensitive to KPT-185 than Nutlin-sensitive cases (12.2 ± 0.06 % versus 30.9 ± 0.04 % Annexin V, P < 0.05). Synergistic induction of apoptosis was not observed in normal cord blood CD34+CD38- cells. Collectively, CRM1 inhibition offers a novel therapeutic strategy for AML that mostly retains wild-type p53. We propose to develop novel combinatorial approaches for the therapy of AML, aimed at maximal activation of p53 and apoptosis signaling by concomitant MDM2 and CRM1 inhibition. Disclosures: Shacham: Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics: Employment. Andreeff:Hoffmann-La Roche: Research Funding; Karyopharm Therapeutics: Unrestricted gift, Unrestricted gift Other.
Glutamine 114 of OYE1 is a well conserved residue in the active site of the Old Yellow Enzyme family. It forms hydrogen bonds to the O2 and N3 of the flavoprotein prosthetic group, FMN. Glutamine 114 was mutated to asparagine, introducing an R-group that is one methylene group shorter. The resultant enzyme was characterized to determine the effect of the mutation on the mechanistic behavior of the enzyme, and the crystal structure was solved to determine the effect of the mutation on the structure of the protein. The Q114N mutation results in little change in the protein structure, moving the amide group of residue 114 out of H-bonding distance, allowing repositioning of the FMN prosthetic group to form new interactions that replace the lost H-bonds. The mutation decreases the ability to bind ligands, as all dissociation constants for substituted phenols are larger than for the wild type enzyme. The rate constant for the reductive half-reaction with -NADPH is slightly greater, whereas that for the oxidative half-reaction with 2-cyclohexenone is smaller than for the wild type enzyme. Oxidation with molecular oxygen is biphasic and involves formation and reaction with O 2 . , a phenomenon that is more pronounced with this mutation than with wild type enzyme. When superoxide dismutase is added to the reaction, we observe a single-phase reaction typical of the wild type enzyme. Turnover reactions using -NADPH with 2-cyclohexenone and molecular oxygen were studied to further characterize the mutant enzyme.
We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.
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