Treatment of B-RAF mutant human tumor cells with a MEK inhibitor requires Bim and is enhanced by a BH3 mimetic
Mark S. Cragg, Elisa S. Jansen, Michele Cook, Claire Harris, Andreas Strasser, Clare L. Scott
Mark S. Cragg, Elisa S. Jansen, Michele Cook, Claire Harris, Andreas Strasser, Clare L. Scott
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Research Article

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-xL 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.

Authors

Mark S. Cragg, Elisa S. Jansen, Michele Cook, Claire Harris, Andreas Strasser, Clare L. Scott

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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.

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Addition of ABT-737 greatly enhances the MEK inhibition–induced apoptosi...
(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.