Bak Binding to Bcl-x_L and Not to Mcl-1 Is Associated with ABT-737 Sensitivity in Multiple Myeloma Cell Lines

ABT-737 is a Bad-like BH3 mimetic and an effective inhibitor of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-x_L and Bcl-w, but not Mcl-1. Recent studies have shown this new drug as a promising anti-cancer agent with activity in multiple myeloma cells. The purpose of this study was to evaluate the role of Bcl-2 family members in both determining the sensitivity and the mechanism of action of ABT-737 in multiple myeloma cell lines. ABT-737, as a single agent, induced apoptosis in six myeloma cell lines, although the sensitivity was quite different among cell lines. Three cell lines 8226/S, MM.1s and KMS18, were highly sensitive to ABT-737 with EC50 values of 0.30, 0.39 and 0.58 μM, respectively. In contrast, three cell lines, KMS11, U266 and OPM2 displayed lower sensitivity to the drug with EC50 values of 1.60, 2.58 and 2.57 μM ABT-737. No correlation between the sensitivity to ABT-737 and the expression pattern of the Bcl-2 family members was found. Interestingly, Mcl-1, a critical anti-apoptotic protein involved in myeloma cell survival that has also been shown to confer resistance to ABT-737, did not correlate with sensitivity to the drug. Bfl-1, an anti-apoptotic Bcl-2 family member with similar functions to Mcl-1, was only expressed in two sensitive cell lines, MM.1s and KMS18. Since the expression pattern did not reveal any strong correlation, we determined the effects of ABT-737 on association of Bcl-2 proteins. Co-immunoprecipitation experiments in MM.1s and KMS11, demonstrated that ABT-737 released Bak and Bim from Bcl-x_L and Bim from Bcl-2 while no change was observed for Bak and Bim bound to Mcl-1. A closer look at the interaction of Bcl-2 family members revealed that Bak is equally bound to Mcl-1 and Bcl-x_L in the less sensitive cell lines while it is primarily bound to Bcl-x_L in the more sensitive cell lines 8226/S and KMS18. Interestingly, Bak in equally bound to Mcl-1 and Bcl-xL in MM.1s, the third sensitive cell line; however, Bim is also highly bound to Bcl-xL, suggesting an easier release of Bak and Bim by ABT-737 from a Bim-primed-Bcl-x_L. Consistent with this idea, Bcl-xL overexpression significantly protected 8226/S but not KMS11 from ABT-737-induced death. Additionally, while silencing of Bim significantly protected MM.1s and KMS11 from ABT-737-induced apoptosis, release of Bak from Bcl-xL was not observed after Bim silencing in the MM.1s cells. Together these data suggest that the interaction pattern not the expression pattern of Bcl-2 proteins is a more accurate measure of ABT-737 function in cells. This is important in diseases like multiple myeloma where Mcl-1 in addition to other anti-apoptotic Bcl-2 proteins are typically expressed.