Treatment of B-RAF mutant human tumor cells with a MEK inhibitor requires Bim and is enhanced by a BH3 mimetic

Abstract

B-RAF is frequently mutated in solid tumors, resulting in activation of the MEK/ERK signaling pathway and ultimately tumor cell growth and survival. MEK inhibition in these cells results in cell cycle arrest and cytostasis. Here, we have shown that MEK inhibition also triggers limited apoptosis of human tumor cell lines with B-RAF mutations and that this effect was dependent on upregulation and dephosphorylation of the proapoptotic, Bcl-2 homology 3-only (BH3-only) Bcl-2 family member Bim. However, upregulation of Bim was insufficient for extensive apoptosis and was countered by overexpression of Bcl-2. To overcome apoptotic resistance, we treated the B-RAF mutant cells both with MEK inhibitors and with the BH3 mimetic ABT-737, resulting in profound synergism and extensive tumor cell death. This treatment was successful because of both efficient antagonism of the prosurvival Bcl-2 family member Mcl-1 by Bim and inhibition of Bcl-2 and Bcl-x(L) by ABT-737. Critically, addition of ABT-737 converted the predominantly cytostatic effect of MEK inhibition to a cytotoxic effect, causing long-term tumor regression in mice xenografted with human tumor cell lines. Thus, the therapeutic efficacy of MEK inhibition requires concurrent unleashing of apoptosis by a BH3 mimetic and represents a potent combination treatment for tumors harboring B-RAF mutations.

Figure 1. MEK inhibition causes growth arrest and apoptosis in B-RAF mutant tumor cells.

(A and B) B-RAF WT (PC3) or mutant (SkMel-28 and Colo205) cells were not treated (NT) or were treated for 16 or 72 h with the MEK inhibitor UO126 (20 μM unless otherwise indicated), and DNA content was determined by FACS analysis. (A) Illustrative FACS plots show untreated cells, cells undergoing G₁ arrest and apoptosis after 16 and 72 h, respectively, of UO126 treatment. Bars denote sub-G₁ DNA content. (B) Percent cells with sub-G₁ DNA content at 72 h. (C) Colo205 cells were treated for 48 h with the indicated doses of UO126 or PD98059. Cells were analyzed by Western blotting for phosphorylated ERK (pERK1/2), total ERK, PARP, cleaved caspase-3, and β-actin as loading control and were also assessed for cell death (shown at right). For B and C, data are mean ± SD of 3 independent experiments. (D) Colo205 cells were not treated or were incubated with 25 μM QVD-OPH (QVD) for 30 min prior to addition of 20 μM UO126 and assessed after 48 h for cell death and cell cycle. Colo205 cells overexpressing FLAG–Bcl-2 were assessed in the same manner. Data are mean ± SD of 3 independent experiments using both FLAG–Bcl-2 clones. (E) Bcl-2 expression levels for clones 1-3 and 1-6. Filled histogram represents staining with a control antibody.

Figure 2. Effect of MEK inhibition on the expression and phosphorylation of BH3-only proteins and prosurvival Bcl-2 family members.

(A) B-RAF WT PC3 cells and B-RAF mutated Colo205 cells were not treated or were treated for 6, 24, or 48 h with 20 μM UO126 and assessed by Western blotting for expression of the indicated proteins. (B) Colo205 cells were treated for 48 h with UO126 and assessed by Western blotting for the indicated proteins. The lysates examined here were the same as those probed in Figure 1C, and the blots shown for phosphorylated ERK, total ERK, and actin are identical, included to allow for direct comparison between loss of ERK phosphorylation and change in apoptosis proteins. Western blot analysis of Bax and Bak levels was performed with new lysates from identically treated cells, with equal loading demonstrated by probing for β-actin. (C) PC3 and Colo205 cells were not treated or were treated for 18 h with 20 μM UO126, harvested, and lysed. Lysates were not treated or were treated with λ phosphatase, and the migration of Bim was assessed by Western blotting. In healthy Colo205 cells, Bim_EL appeared as a broad band (arrow). Treatment with λ phosphatase produced a single band of apparent lower molecular weight similar to that after treatment with UO126. (D) Control and Bcl-2–overexpressing Colo205 cells were not treated or were treated for 6, 24, or 48 h with 20 μM UO126 and assessed by Western blotting. Data are representative of 3 independent experiments.

Figure 3. MEK inhibition–induced apoptosis of B-RAF mutant tumor cells can be inhibited by Bim KD or Bcl-2 overexpression.

(A) Top: Western blot analysis documents the levels of Bim and β-actin (loading control) expression in parental and 2 independent RNAi Bim KD subclones of Colo205 cells. Bottom: Parental and RNAi Bim KD subclone 18 Colo205 cells were not treated or were treated for 6 or 24 h with 20 μM UO126 and analyzed by Western blotting for their levels of Bim. (B) Parental, Bim RNAi KD, and Bcl-2–overexpressing clones of Colo205 cells were treated for 48 h with 0–40 μM UO126 as indicated, and cell survival was examined by FACS analysis. Data (mean ± SD of 3 or 4 independent clones) indicate percent cell death relative to untreated cells. (C) Clonogenic survival assays of parental, Bim RNAi KD, and Bcl-2–overexpressing clones of Colo205 cells without treatment or after 24 or 48 h of treatment with 20 μM UO126. Data are mean ± SD of 3 independent experiments.

Figure 4. MEK inhibition causes induction and dephosphorylation of Bim in a range of B-RAF mutant tumor cells.

(A) MM200-1, SkMel-28, Mel-RMU, and MCF-7 tumor-derived cell lines were not treated, were treated with 20 μM UO126 for the indicated time points, or were treated with the indicated concentrations of UO126 for 18 h, and were assessed for levels of Bim, phosphorylated ERK1/2, and total ERK1/2 by Western blotting. D, DMSO control. (B) SkMel-28 cells were not treated or were treated for 18 h with 20 μM UO126, harvested, and lysed. Lysates were not treated or were treated with λ phosphatase and then assessed by Western blotting for the migration of Bim on SDS-PAGE. Arrow indicates the faint diffuse band of Bim present in untreated healthy cells. (C) Untreated PC3, MM200-1, SkMel-28, Mel-RMU, and Colo205 cells were assessed by Western blotting for the indicated apoptosis-related proteins, all on the same membranes to allow direct comparisons.

Figure 5. Addition of ABT-737 greatly enhances the MEK inhibition–induced apoptosis of B-RAF mutant tumor cells by increasing the binding of Bim to Mcl-1.

(A and B) Colo205 cells were treated with ABT-737 plus 0 or 20 μM UO126 (A) or with UO126 plus 0 or 1 μM ABT-737 (B). Cell killing was assessed after 48 h as described in Figure 3B. (C) Bim RNAi KD or Bcl-2–overexpressing Colo205 cells were treated with 0 or 1 μM ABT-737 plus 0 or 20 μM UO126, and cell killing was assessed after 48 h. Data show percent apoptosis compared with untreated cells. (D) MM200-1, SkMel-28, PC3, or MCF-7 cells were not treated or were treated with 20 μM UO126, 1 μM ABT-737, or both, and cell killing was assessed after 48 h. For A–D, data represent mean ± SD of 3 independent experiments. (E) Colo205 or Colo205–Bcl-2 cells were not treated or were treated for 18 h with 1 μM ABT-737 (A), and lysates were subjected to anti-Bim immunoprecipitation and Western blot analysis. (F) Colo205 cells were treated for 18 h with 20 μM UO126 (UO) in the presence or absence of 1 μM ABT-737. Lysates were subjected to anti-Bim immunoprecipitation and Western blot analysis. (G) CBA nu/nu mice were inoculated with Colo205 tumor cells; when tumors were palpable, mice were treated with 75 mg/kg ABT-737 daily for 2 d. Tumors were dissected 48 h later, and lysates were subjected to anti-Bim immunoprecipitation and Western blotting.

Figure 6. ABT-737 enhanced the therapeutic effect of PD0325901 in the treatment of B-RAF mutant tumor–bearing nude mice.

(A) CBA nu/nu mice were inoculated with SkMel-28 tumor cells; when tumors reached the target size of 0.3 cm³, mice were treated with PD0325901 (P; 3 mg/kg body weight), ABT-737 (75 mg/kg body weight), both drugs (A+P), or vehicle (V) daily for 2 d. Tumors were then dissected, and cell lysates were subjected to Western blot analysis with antibodies to Bim. (B and C) SkMel-28 tumor cells were inoculated into CBA nu/nu mice; once tumors reached the target size of 0.1 cm³, mice were treated once daily for 10 consecutive d with PD0325901, ABT-737, both drugs, or vehicle. (B) Representative tumors from C at the time of first treatment and at time of cull of the first tumor-bearing mice (vehicle treated). (C) Average tumor size, measured throughout and represented as the percentage of tumor size at the time treatment began. n = 10–12 mice per treatment group. Data are mean ± SD.