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. 2012 Apr;26(4):778-87.
doi: 10.1038/leu.2011.287. Epub 2011 Nov 8.

MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex

M Konopleva  1 , M Milella, P Ruvolo, J C Watts, M R Ricciardi, B Korchin, T McQueen, W Bornmann, T Tsao, P Bergamo, D H Mak, W Chen, J McCubrey, A Tafuri, M Andreeff
Affiliations

MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex

M Konopleva et al. Leukemia. 2012 Apr.

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Abstract

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

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Conflict of interest statement

Conflict of Interest Statement: There is no conflict of interest to disclose.

Figures

Figure 1
Figure 1. ABT-737 and PD0325901 are synergistic in leukemic cell lines
A) OCI-AML3 cells were cultured in the presence of escalating doses of ABT-737 (10, 100, 250, 500, 1000 nM), PD0325901 (10, 100, 250, 500, 1000 nM), or the combination of the 2 agents at a fixed ratio added simultaneously. After 48 hours, apoptosis was measured by annexin V flow cytometry. Results are expressed as means ± SEM of the results of 3 replicates. (B) CI values were calculated using the Calcusyn software. CI values of less than 1.0 indicate synergism. Annexin V CI values are shown for OCI-AML3, HL60, and MOLM13 cell lines. ABT-737 and PD0325901 doses used for HL60: 10, 100, 250, and 500 nM for both drugs. Doses used for MOLM13: ABT-737 (10, 100, 250, 500, 1000 nM) and PD0325901 (1, 10, 25, 50, 100 nM).
Figure 2
Figure 2. PD0325901 reduces MCL-1 protein levels and alters Bim phosporylation
A) OCI-AML3 cells were treated with 10, 100, or 500 nM of ABT-737 and/or PD0325901 for 24 hours. Levels of pERK and ERK2 were analyzed by immunoblot. Ratio of p-ERK/ERK was determined by densitometry of bands using Image J software. B) MOLM13 cells were treated with 40 nM of ABT-737 and/or 80 nM PD0325901 for 24 hours. Levels of pERK and ERK were analyzed by immunoblot. Ratio of p-ERK/ERK was determined by densitometry of bands using Image J software. C) OCI-AML3 cells were treated with 100 nM of ABT-737 and/or PD0325901 for 6, 12, or 24 hours. Levels of MCL-1, and GAPDH were analyzed by immunoblot. Ratio of MCL-1/GAPDH was determined by densitometry of bands using Image J software.
Figure 3
Figure 3. ABT-737 and PD0325901 treatment activates Bak and increases the association of MCL-1 with Bim
A) OCI-AML3 cells were treated with ABT-737 and/or PD0325901 for 6 hours. Cell lysates were immunoprecipitated with an antibody to activated Bak (Calbiochem), and then immunoblotted for total Bak. B) OCI-AML3 cells were treated with ABT-737 and/or PD0325901 for 1 hour. Cell lysates were immunoprecipiated with an antibody to MCL-1 (Santa Cruz), and then immunoblotted for Bim and Bak.
Figure 4
Figure 4. Loss of Bim and/or Bak expression partially protects cells from ABT-737 and PD0325901 induced apoptosis
A) MEF cells with Bak, Bax, Bak and Bax, or Bim expression knocked out were treated for 48 hours with 5 uM ABT-737 and/or PD0325901. Apoptosis was assessed by measuring annexinV by flow cytometry. Results are expressed as means ± SEM of the results of 3 replicates. B) Bak, Bax, and Bim immunoblots of the MEF cells lines. C) OCI-AML3 cells were Amaxa transfected with Bim siRNA and/or Bak ON-TARGETplus SMARTpool siRNA. Cells were treated with 100 nM ABT-737 and/or PD0325901 24 hours after transfection, and after 24 hours of drug treatment, apoptosis was assessed by annexinV flow cytometry. Results are expressed as means ± SEM of the results of 3 replicates. D) Bak and Bim immunoblots showing the extent of protein level reduction by siRNA. Bands were quantitated using Scion Image software and gel loading differences were corrected for using the matching gapdh immunoblot. The DMSO condition was set to 1.
Figure 5
Figure 5. MEK inhibitors promote ABT-737-induced killing of primary AML blast cells
A) AML blast cells were cultured in the presence of escalating doses of 100 nM ABT-737, 1 μM CI1040, or the combination of the 2 agents. After 48 hours, apoptosis was measured by annexin V flow cytometry. B) Induction of apoptosis by ABT-737 and CI1040 (doses as in A) in 8 primary AML samples (same as in A) and in three samples of CD34+ cells isolated from normal apheresis samples. Results are expressed as the mean ± SEM. C) Effects of ABT-737 and PD0325901 on AML stem cells. The frequency of CD34+CD38−CD123+ Annexin V+ cells was determined by multicolor flow cytometry. The percentage of non-apoptotic [annexin V(−)] stem cells was calculated after ABT-737 and PD0325901 treatment (number of stem cells in DMSO-treated cultures = 100%). Results are expressed as the mean ± SEM.
Figure 6
Figure 6. In vivo effects of combined BCL2/BCL-XL (ABT-737) and MEK (CI-1040) inhibition in Molm-13-transplanted nude mice
A) Human leukemia luciferase-expressing Molm-13 cells were injected IV into nu/nu mice, and leukemia dissemination was monitored by bioluminescence imaging. Two weeks post cell injection mice were treated with ABT-737, CI-1040 or combination (n=8 per group). Serial images of mice are shown. B) Results were averaged from the peak light-emitting exposure from each group and displayed as photons/sec.
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