BH3 mimetic drugs may be useful to treat acute lymphoblastic leukemia (ALL) but the sensitivity of primary tumor cells has not been fully evaluated. Here B-lineage ALL cell cultures derived from a set of primary tumors were studied with respect to sensitivity to the BH3 mimetics ABT-263 and ABT-199 and to Bcl-2 dependence and function. These ALL cells each expressed high levels of Bcl-2 and exhibited great sensitivity to ABT-263 and ABT-199, which induced rapid apoptotic cell death. BH3 profiling indicated that the ALL cultures were Bcl-2 dependent. Co-immunoprecipitation studies revealed a multi-faceted role for Bcl-2 in binding pro-apoptotic partners including Bax, Bak, Bik and Bim. ABT-263 disrupted Bcl-2:Bim interaction in cells. Mcl-1 overexpression rendered ALL cells resistant to ABT-263 and ABT-199 with Mcl-1 assuming the role of Bcl-2 in binding Bim. Freshly isolated pediatric ALL blasts also expressed high levels of Bcl-2 and exhibited high sensitivity to Bcl-2 inhibition by the BH3 mimetic compounds. Overall our results showed that primary ALL cultures were both more sensitive to BH3 mimetics and more uniform in their response than established ALL cell lines which have been evaluated previously. Further, the primary cell model characterized here offers a powerful system for preclinical testing of novel drugs and drug combinations to treat ALL.

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.

Background: The molecular basis of variable cell fate after mitotic arrest is poorly understood. Results: The robustness of Cdk1 signaling to antiapoptotic Bcl-2 proteins dictates mitotic death versus mitotic slippage. Conclusion: Sustained Cdk1 activity coordinately promotes mitotic arrest and mitotic death. Significance: Defining the molecular basis of antimitotic drug action is important for their rational use clinically.

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.

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