BAK complex

Abstract: We previously reported that hyperforin, a phloroglucinol purified from Hypericum perforatum, induces the mitochondrial pathway of caspase-dependent apoptosis in chronic lymphocytic leukemia (CLL) cells ex vivo, and that this effect is associated with upregulation of Noxa, a BH3-only protein of the Bcl-2 family. Here, we investigated the role of this upregulation in the pro-apoptotic activity of hyperforin in the cells of CLL patients and MEC-1 cell line. We found that the increase in Noxa expression is a time- and concentration-dependent effect of hyperforin occurring without change in Noxa mRNA levels. A post-translational regulation is suggested by the capacity of hyperforin to inhibit proteasome activity in CLL cells. Noxa silencing by siRNA reduces partially hyperforin-elicited apoptosis. Furthermore, treatment with hyperforin, which has no effect on the expression of the prosurvival protein Mcl-1, induces the interaction of Noxa with Mcl-1 and the dissociation of Mcl-1/Bak complex, revealing that upregulated Noxa displaces the proapoptotic protein Bak from Mcl-1. This effect is accompanied with Bak activation, known to allow the release of apoptogenic factors from mitochondria. Our data indicate that Noxa upregulation is one of the mechanisms by which hyperforin triggers CLL cell apoptosis. They also favor that new agents capable of mimicking specifically the BH3-only protein Noxa should be developed for apoptosis-based therapeutic strategy in CLL.

Abstract: Mcl-1, a member of the Bcl/Mcl family, is a key protein involved in evasion of apoptosis in a wide variety of tumors. Its amplification and overexpression have also been implicated in innate and acquired resistance to anticancer drugs. Mcl-1 is capable of preventing induction of apoptosis, both by binding and inactivating the pro-apoptotic executioner Bcl-2 protein, Bak, as well as by sequestering other pro-apoptotic BH3-only proteins such as Bim and Noxa. AZD5991 is a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1. It demonstrates all the hallmarks of a true Mcl-1 inhibitor: 1. potent, selective, and rapid apoptosis in Mcl-1-dependent cell lines (e.g., GI50 as low as 10 nM in multiple myeloma cell lines); 2. loss of activity upon overexpression of Bcl-xL or siRNA-mediated knockout of Bak; 3. Mcl-1:Bak complex disruption as demonstrated by co-immunoprecipitation. AZD5991 is active in vivo, with complete (100%) tumor regression demonstrated in several mouse xenograft models after a single tolerated dose. We have also demonstrated synergistic in vivo efficacy in combination with standard-of-care agents. Analysis of ex vivo activity in primary samples from leukemia patients indicates that a high percentage of leukemia patients should respond to drug treatment, which supports our plan for a phase I trial of AZD5991 in patients with hematologic cancers. Citation Format: Alexander W. Hird, J. Paul Secrist, Ammar Adam, Matthew A. Belmonte, Eric Gangl, Frank Gibbons, David Hargreaves, Jeffrey W. Johannes, Stephen L. Kazmirski, Jason G. Kettle, Stephen E. Kurtz, Michelle L. Lamb, Martin J. Packer, Bo Peng, Craig R. Stewart, Jeffrey W. Tyner, Wenzhan Yang, Qing Ye, XiaoLan Zheng, Edwin A. Clark. AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT01-02. doi:10.1158/1538-7445.AM2017-DDT01-02

Abstract: Although the pro-apoptotic BCL-2 family proteins BAK and BAX play a key role in mitochondrial perturbation their transition from an inactive closed conformation to a membrane permeabilising pore remains unclear. I found that BAK in viable cells existed in a primed state which was characterized by an occluded N-terminus and an exposed BH3 domain. This conformation facilitated binding to the hydrophobic groove of BCL-XL and served as a checkpoint maintaining cell survival by preventing its further activation. Isolation of BAK by immunoprecipitation suggests that only a discrete portion is present in this primed conformation. Reconstitution of the BCL-XL BAK complex into a BAK/BAX null background rendered cells more sensitive to the BAD BH3 mimetic ABT-737 indicating that primed BAK is primarily involved in ABT-737 induced apoptosis. Primed BAK was displaced from BCL-XL by ABT-737 followed by an N-terminal conformational change and subsequent formation of dimers and higher molecular weight complexes. These sequential BAK activation steps occurred independently of cell fate and did not represent the rate limiting steps in BAK activation as a BAK BH3 mutant L78A lost proapoptotic function but still oligomerised as efficiently as wt BAK. Thus the transition from inactive BAK to a membrane permeabilising pore requires an additional activation step. I demonstrate that after 30 min of ABT-737 exposure primed BAK, after its displacement from BCL-XL, interacts with BIMEL reflecting the transient nature of this interaction. This interaction represented an additional step in BAK activation as BAK pro-apoptotic function was enhanced when BIMEL and BAK were co-expressed. However BIMEL did not induce the N-terminal conformational change nor oligomerisation of BAK and its interaction occurred downstream of both these events. In addition the pool of BIMEL involved in the further activation of BAK did not represent that sequestered by the antiapoptotic proteins BCL-2 and BCL-XL. These data suggest that BAK activation occurs in multiple steps in which a further activation event is required after the exposure of the BH3 domain, the N-terminal conformational change and the formation of high molecular weight complexes but prior to cytochrome c release. I propose that this event may be represented by interaction of N-terminal conformational changed/oligomerised BAK with BIMEL. Acknowledgments I thank my superisor Prof. Gerry Cohen. I am grateful to all members of the lab both past and present and especially to Dr. Nicholas Harper for his helpful discussions and experimental support. I also thank Prof. John Schwab for support in designing the mutants and disussions. Ausserdem mochte ich ganz lieb meiner Familie danken. Ganz besonders meiner kleinen Hanni ohne deine ansteckende Lebensfreude hatte ich es nie geschafft. Ausserdem Nick vielen Dank, dass du immer fur mich da warst und bist und alle Hohen und Tiefen mit mir meisterlich durchgestanden hast. Zudem ein riesengrosses Dankeschon an meine Eltern fur eure kontinuierliche Unterstutzung, ohne Euch ware es nie moglich gewesen. Danke, dass ihr immer fur uns da wart.