MDM2 Inhibitor, Nutlin 3a, Induces p53 Dependent Autophagy in Acute Leukemia by AMP Kinase Activation.

MDM2 (mouse double minute 2) inhibitors that activate p53 and induce apoptosis in a non-genotoxic manner are in clinical development for treatment of leukemias. P53 can modulate other programmed cell death pathways including autophagy both transcriptionally and non-transcriptionally. We investigated autophagy induction in acute leukemia by Nutlin 3a, a first-in-class MDM2 inhibitor. Nutlin 3a induced autophagy in a p53 dependent manner and transcriptional activation of AMP kinase (AMPK) is critical, as this effect is abrogated in AMPK -/- mouse embryonic fibroblasts. Nutlin 3a induced autophagy appears to be pro-apoptotic as pharmacological (bafilomycin) or genetic inhibition (BECLIN1 knockdown) of autophagy impairs apoptosis induced by Nutlin 3a.

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells.

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.

Prognostic impact and targeting of CRM1 in acute myeloid leukemia.

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(+)/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.

Decreased sensitivity of 17p-deleted chronic lymphocytic leukemia cells to a small molecule BCL-2 antagonist ABT-737.

BACKGROUND: Despite the high complete response rates achieved with fludarabine-based regimens, relapse is inevitable in chronic lymphocytic leukemia (CLL). Relapsed patients often acquire deletions of the short arm of chromosome 17 (del[17p]), which are closely associated with tumor protein 53 (TP53) mutations. Wild-type p53 up-regulates and activates B-cell CLL/lymphoma 2 (BCL-2)-associated X protein (BAX), and it down-regulates and inactivates BCL-2. The small-molecule BCL-2 inhibitor ABT-737 induces apoptosis in a BAX-dependent and BCL-2 homologous antagonist-killer (BAK)-dependent manner. The role of p53 in sensitivity of CLL cells to BCL-2 inhibition has not been extensively investigated. METHODS: The authors investigated the association of del(17p) with ABT-737 sensitivity in CLL cells from 50 patients. Stable p53 and BAX knockdown cells were used for mechanistic studies. RESULTS: CLL cells with del(17p) were less sensitive to ABT-737-induced BAX activation and apoptosis than CLL cells without del(17p) (39% ± 7.3% vs 63.7% ± 2.9% [specific annexin V induction]; P < .01). A positive correlation between the degrees of apoptosis induced by ABT-737 and by the p53-activating binding protein homolog murine double minute (MDM2) antagonist nutlin-3a (correlation coefficient [r] = 0.75; P < .0001) was observed. CLL cells with del(17p) expressed lower levels of BAX than those without del(17p) (0.67 ± 0.12 vs 1.27 ± 0.10 in relative protein expression levels; P < .01). Knockdown of p53 or BAX in leukemia cells resulted in decreased apoptosis induced by ABT-737. CONCLUSIONS: The current data indicated that p53 dysfunction may lead to decreased apoptosis induction by ABT-737.

Activation of p53 signaling by MI-63 induces apoptosis in acute myeloid leukemia cells.

Non-mutational inactivation of p53 is frequent in acute myeloid leukemia (AML) via overexpression of MDM2. We report that treatment with MI-63, a novel inhibitor of MDM2, activates p53 signaling to induce apoptosis in AML cell lines and primary samples. Cell lines naturally devoid of p53 or expressing shRNA targeting p53 are refractory to apoptosis induction by MI-63, indicating that the effects of MI-63 require p53 expression. MI-63 induced G1 phase arrest and increased p21 expression. MI-63 induced pronounced apoptosis in all primary AML samples tested, and most important, was effective in inducing cell death of leukemia 'stem' cells. In addition, MI-63 showed synergy with both doxorubicin and AraC. Interestingly, treatment with MI-63 also led to a reduction in levels of MDM4 protein, a repressor of p53 mediated transcription, in AML cells. Our results warrant investigation of MI-63 or its analogs as anti-leukemic agents, alone or in combination with traditional chemotherapeutic agents.

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

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.

The role of glutamine 114 in old yellow enzyme.

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 beta-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 beta-NADPH with 2-cyclohexenone and molecular oxygen were studied to further characterize the mutant enzyme.