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. 2014 Jul;13(7):1848-59.
doi: 10.1158/1535-7163.MCT-13-0576. Epub 2014 Apr 16.

"V体育官网" Evaluation of apoptosis induction by concomitant inhibition of MEK, mTOR, and Bcl-2 in human acute myelogenous leukemia cells

Weiguo Zhang  1 , Vivian R Ruvolo  1 , Chen Gao  1 , Liran Zhou  1 , William Bornmann  2 , Twee Tsao  1 , Wendy D Schober  1 , Paul Smith  3 , Sylvie Guichard  3 , Marina Konopleva  4 , Michael Andreeff  4
Affiliations

Evaluation of apoptosis induction by concomitant inhibition of MEK, mTOR, and Bcl-2 in human acute myelogenous leukemia cells

"VSports最新版本" Weiguo Zhang et al. Mol Cancer Ther. 2014 Jul.

Abstract

Aberrant activation of multiple signaling pathways is common in acute myelogenous leukemia (AML) cells, which can be linked to a poor prognosis for patients with this disease. Previous research with mTOR or MEK inhibitors revealed cytostatic, rather than cytotoxic, effects in in vitro and in vivo AML models. We evaluated the combination effect of the mTOR inhibitor AZD8055 and the MEK inhibitor selumetinib on human AML cell lines and primary AML samples VSports手机版. This combination demonstrated synergistic proapoptotic effects in AML cells with high basal activation of MEK and mTOR. We next incorporated the BH3 mimetic ABT-737 into this combination regimen to block Bcl-2, which further enhanced the apoptogenic effect of MEK/mTOR inhibition. The combination treatment also had a striking proapoptotic effect in CD33(+)/CD34(+) AML progenitor cells from primary AML samples with NRAS mutations. Mechanistically, upregulation of the proapoptotic protein Bim, accompanied by the downregulation of the antiapoptotic protein Mcl-1 (mainly via protein degradation), seemed to play critical roles in enhancing the combination drug effect. Furthermore, the modulation of survivin, Bax, Puma, and X-chromosome-linked inhibitor of apoptosis protein (XIAP) expression suggested a role for mitochondria-mediated apoptosis in the cytotoxicity of the drug combination. Consequently, the concomitant blockade of prosurvival MEK/mTOR signaling and the deactivation of Bcl-2 could provide a mechanism-based integrated therapeutic strategy for the eradication of AML cells. .

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Figures

Figure 1
Figure 1
Dual blockade of mTOR/MEK signaling synergistically or additively inhibits growth in AML lines OCI/AML3, MOLM13, and U937 but not KG-1. A, AML cells were treated with AZD8055, selumetinib, or both at the indicated concentrations for 48 hours. Inhibition of cell growth was measured using viable cell counts as described in Materials and Methods and expressed as the percentage of untreated cells. Data shown represent the mean of four independent determinations. Error bars correspond to 95% confidence intervals. *P < 0.05; ** P < 0.01. B, Basal expression levels of phosphorylated and total ERK, AKT and Bcl-2 proteins was determined by immunoblotting. Numbers indicate ratio of phosphorylated proteins to the respective total proteins. C, AML cells were treated with AZD8055 and selumetinib at the indicated concentrations for 24 hours, and the profile of signaling and cell-cycle–related checkpoint proteins was measured by immunoblotting. α-tubulin was used as loading control. D, AML cells were treated with the indicated agents for 24 hours, and BrdU incorporation was determined using flow cytometry after labeling with anti-BrdU-fluorescein isothiocyanate antibody. Data represent the mean of triplicated experiments. DMSO, dimethyl sulfoxide control.
Figure 2
Figure 2
Synergistic proapoptotic effects induced by blockade of mTOR/MEK signaling are associated with modulation of Mcl-1 and Bim. A, AML cells were treated with AZD8055 (0.3 μmol/L), selumetinib (0.9 μmol/L), or both for 72 hours, and the percentage of apoptotic cells was determined using flow cytometry. B, Bcl-2 family proteins were assessed by immunoblotting after 24 hours of treatment with AZD8055 and selumetinib. The numbers below the blots indicate the ratio of protein to the loading control β-actin. C, OCI/AML3 and MOLM13 cells were treated with indicated inhibitors for 6 hours, and relative mRNA expression levels were determined by real-time PCR by calculating the ratios of Mcl-1/Bim to 18S mRNA. D, OCI/AML3 cells were treated for 48 hours in the presence or absence of cycloheximide (0.5 μmol/L), and apoptosis was determined by flow cytometry. Inset: protein levels of Survivin, Mcl-1, and Bim after treatment of OCI/AML3 cells with cycloheximide (CHX) for 6 hours. *P < 0.05; ** P < 0.01; *** P < 0.001; NS; no statistical significance.
Figure 3
Figure 3
Levels of proapoptotic BH3-only Bim protein and antiapoptotic Mcl-1 protein play a critical role in drug combination-induced apoptosis. A, OCI/AMl3 cells were electroporated with Bim siRNA or scrambled siRNA for 24 hours, treated with AZD8055 (0.4 μmol/L) and/or selumetinib (0.2 μmol/L) for 48 hours, and examined for apoptosis induction. Inset: knockdown of basal level of Bim protein expression. B, OCI/AML3 cells were transferred with Mcl-1 shRNA or shGFP control lentiviral vectors, the puromycin-selected cells were treated with AZD8055 (0.4 μmol/L) and/or selumetinib (0.4 μmol/L) for 48 hours, and examined for apoptosis induction. Inset: knockdown of basal level of Mcl-1 after puromycin selection. DMSO, dimethyl sulfoxide, control. ** P < 0.01.
Figure 4
Figure 4
ABT-737 further enhances the pro-apoptotic effect of the AZD8055/selumetinib combination treatment in AML cells. A, AML cells were treated with AZD8055, selumetinib, ABT-737, or the indicated combinations for 48 hours, and examined for apoptosis induction. B, U937 cells were treated as described above and examined for apoptosis induction. ** P < 0.01; *** P < 0.001. C, KG-1 and OCI/AML3 cells were treated with ABT-737, AZD8055 plus selumetinib, or both at the indicated concentrations (in μmol/L) for 6 hours, and Bcl-2 protein was pulled down with anti-Bcl-2 antibody-conjugated A/G beads. Bcl-2–binding Bax protein expression was measured using immunoblotting. A 10% Bcl-2 input of immunoprecipitated fractions is shown as a loading control for Bcl-2 protein level. IP, immunoprecipitation; WB, immunoblot. D, KG-1 and OCI/AML3 cells were treated with ABT-737, AZD8055 plus selumetinib, or both for 24 hours, and the cells were lysed to assess expression of apoptosis-related Bcl-2 family members in mitochondrial, cytosol, and whole-cell fractions via immunoblotting. Casp, caspase; EL, extra-long; L, long and S, short.
Figure 5
Figure 5
Three-drug combination treatment exerts synergistic proapoptotic effects in AML blasts with NRAS mutations. Mononuclear cells obtained from peripheral blood and bone marrow samples of AML patients with (A) or without (B) NRAS mutations were exposed to AZD8055, selumetinib, and/or ABT737 at the indicated concentrations (in μmol/L) for 24 to 72 hours. C, The specific apoptosis was determined in triplicate samples based on the triple-drug treatment data shown in Fig. 5A and 5B, and shown as a comparison between bulk and CD33+/34+ fractions of the NRAS mutated and NRAS-wild samples. ** P < 0.01. D, AML patient sample (case 7) was treated with indicated concentration (μmol/L) of inhibitors for 24 hours, and correlative protein expression was determined by immunoblotting.
Figure 6
Figure 6
Mutated NRAS in AML cells are sensitive to selumetinib-induced apoptosis in the presence of ABT-737. A, MV4-11 cells with or without NRAS mutation were treated with AZD8055, selumetinib, or ABT-737, their combinations at the indicated concentrations (μmol/L) for 48 hours, and examined for apoptosis induction. Inset: basal phosphorylation level of p-MEK in wild-type (WT) and NRAS-mutated MV4-11 cells. *P < 0.05; ** P < 0.01. B, NRAS-wild type, and NRAS-mutant MV4-11 cells were treated with the indicated drugs (μmol/L) for 16 hours, and corresponding protein expression was measured by immunoblotting. C, NRAS-WT and –mutated MV4-11 cells were treated with the indicated drugs (μmol/L) for 24 hours, and loss of ΔΨm was evaluated using flow cytometry after staining with CMXRos and MitoTracker Green. ** P < 0.01.
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