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 2

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

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Effect of MEK inhibition on the expression and phosphorylation of BH3-on...
(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, BimEL 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.