Denis E. Reyna

BH3-Triggered Structural Reorganization Drives the Activation of Proapoptotic BAX

BAX is a proapoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α helices 1 and 2 is displaced, the carboxy-terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an autoactivating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway.

Bax regulates primary necrosis through mitochondrial dynamics

The defining event in apoptosis is mitochondrial outer membrane permeabilization (MOMP), allowing apoptogen release. In contrast, the triggering event in primary necrosis is early opening of the inner membrane mitochondrial permeability transition pore (mPTP), precipitating mitochondrial dysfunction and cessation of ATP synthesis. Bcl-2 proteins Bax and Bak are the principal activators of MOMP and apoptosis. Unexpectedly, we find that deletion of Bax and Bak dramatically reduces necrotic injury during myocardial infarction in vivo. Triple knockout mice lacking Bax/Bak and cyclophilin D, a key regulator of necrosis, fail to show further reduction in infarct size over those deficient in Bax/Bak. Absence of Bax/Bak renders cells resistant to mPTP opening and necrosis, effects confirmed in isolated mitochondria. Reconstitution of these cells or mitochondria with wild-type Bax, or an oligomerization-deficient mutant that cannot support MOMP and apoptosis, restores mPTP opening and necrosis, implicating distinct mechanisms for Bax-regulated necrosis and apoptosis. Both forms of Bax restore mitochondrial fusion in Bax/Bak-null cells, which otherwise exhibit fragmented mitochondria. Cells lacking mitofusin 2 (Mfn2), which exhibit similar fusion defects, are protected to the same extent as Bax/Bak-null cells. Conversely, restoration of fused mitochondria through inhibition of fission potentiates mPTP opening in the absence of Bax/Bak or Mfn2, indicating that the fused state itself is critical. These data demonstrate that Bax-driven fusion lowers the threshold for mPTP opening and necrosis. Thus, Bax and Bak play wider roles in cell death than previously appreciated and may be optimal therapeutic targets for diseases that involve both forms of cell death.

An interconnected hierarchical model of cell death regulation by the BCL-2 family

Multidomain pro-apoptotic BAX and BAK, once activated, permeabilize mitochondria to trigger apoptosis, whereas anti-apoptotic BCL-2 members preserve mitochondrial integrity. The BH3-only molecules (BH3s) promote apoptosis by either activating BAX–BAK or inactivating anti-apoptotic members. Here, we present biochemical and genetic evidence that NOXA is a bona fide activator BH3. Using combinatorial gain-of-function and loss-of-function approaches in Bid−/−Bim−/−Puma−/−Noxa−/− and Bax−/−Bak−/− cells, we have constructed an interconnected hierarchical model that accommodates and explains how the intricate interplays between the BCL-2 members dictate cellular survival versus death. BID, BIM, PUMA and NOXA directly induce stepwise, bimodal activation of BAX–BAK. BCL-2, BCL-XL and MCL-1 inhibit both modes of BAX–BAK activation by sequestering activator BH3s and ‘BH3-exposed’ monomers of BAX–BAK, respectively. Furthermore, autoactivation of BAX and BAK can occur independently of activator BH3s through downregulation of BCL-2, BCL-XL and MCL-1. Our studies lay a foundation for targeting the BCL-2 family for treating diseases with dysregulated apoptosis.

An Autoinhibited Dimeric Form of BAX Regulates the BAX Activation Pathway.

Pro-apoptotic BAX is a cell fate regulator playing an important role in cellular homeostasis and pathological cell death. BAX is predominantly localized in the cytosol, where it has a quiescent monomer conformation. Following a pro-apoptotic trigger, cytosolic BAX is activated and translocates to the mitochondria to initiate mitochondrial dysfunction and apoptosis. Here, cellular, biochemical, and structural data unexpectedly demonstrate that cytosolic BAX also has an inactive dimer conformation that regulates its activation. The full-length crystal structure of the inactive BAX dimer revealed an asymmetric interaction consistent with inhibition of the N-terminal conformational change of one protomer and the displacement of the C-terminal helix α9 of the second protomer. This autoinhibited BAX dimer dissociates to BAX monomers before BAX can be activated. Our data support a model whereby the degree of apoptosis induction is regulated by the conformation of cytosolic BAX and identify an unprecedented mechanism of cytosolic BAX inhibition.

Direct Activation of BAX by BTSA1 Overcomes Apoptosis Resistance in Acute Myeloid Leukemia

The BCL-2 family protein BAX is a central mediator of apoptosis. Overexpression of anti-apoptotic BCL-2 proteins contributes to tumor development and resistance to therapy by suppressing BAX and its activators. We report the discovery of BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site and induces conformational changes to BAX leading to BAX-mediated apoptosis. BTSA1-induced BAX activation effectively promotes apoptosis in leukemia cell lines and patient samples while sparing healthy cells. BAX expression levels and cytosolic conformation regulate sensitivity to BTSA1. BTSA1 potently suppressed human acute myeloid leukemia (AML) xenografts and increased host survival without toxicity. This study provides proof-of-concept for direct BAX activation as a treatment strategy in AML.

Progress in targeting the BCL-2 family of proteins

The network of protein-protein interactions among the BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Anti-apoptotic BCL-2 proteins are considered promising targets for drug discovery and exciting clinical progress has stimulated intense investigations in the broader family. Here, we discuss recent developments in small molecules targeting anti-apoptotic proteins and alternative approaches to targeting BCL-2 family interactions. These studies advance our understanding of the role of BCL-2 family proteins in physiology and disease, providing unique tools for dissecting these functions. The BCL-2 family of proteins is a prime example of targeting protein-protein interactions and further chemical biology approaches will increase opportunities for novel targeted therapies in cancer, autoimmune and aging-associated diseases.

Self-regulation of BAX-induced cell death

Apoptosis, a form of programmed cell death, is a process in multicellular organisms responsible for normal tissue development and homeostasis. The intrinsic pathway of apoptosis is principally regulated by protein-protein interactions within the BCL-2 family of proteins, which can prevent or promote mitochondrial dysfunction. There are over twenty BCL-2 family proteins grouped together based on their functional and structural similarities. Specifically, family members are divided into anti-apoptotic and pro-apoptotic proteins and posses up to four BCL-2 Homology (BH) domains, namely BH1, BH2, BH3 and BH4. The BH3 domain is the most conserved and serves as the key domain to mediate protein-protein interactions between the members. Upon a variety of apoptotic stimuli, pro-apoptotic protein BAX triggers the intrinsic apoptotic pathway by inducing mitochondrial outer membrane permeabilization and the release of soluble factors important in caspase activation that are required for apoptosis. The precise mechanisms of BAX activation and inhibition are essential to the understanding of the mitochondrial cell death pathway in physiological and pathological states. Pro-apoptotic BAX resides primarily in the cytosol in a conformation that requires activation for inducing apoptosis [1]. Interaction of cytosolic BAX with the BH3 domain of BH3-only proteins, such as BIM, induces structural conformational changes on BAX enabling it to translocate to the mitochondria and homo-oligomerize into a deadly membrane pore [2, 3]. Specifically, structural studies have identified that BH3-binding to the N-terminal activation site stimulates conformational changes leading to the release of the C-terminal helix α9 from the hydrophobic groove, which facilitates mitochondrial anchoring and oligomerization of BAX [4, 5]. Therefore, two structural regions critical to BAX activation are available for BAX modulation in the cytosol. Because BAX activation requires the availability of its activators, it is not understood if and how BAX can regulate its activation in the presence of apoptotic insults. In search of additional mechanisms that keep cytosolic BAX inactive and regulate its activation, we investigated the hypothesis that other proteins may interact with BAX in the cytosol. For several cell lines that are under non-apoptotic stress, we isolated cytosolic fractions Commentary: Autophagy and Cell Death

Pulling the BAX trigger for tumor cell death

Cancer cells evade cellular death pathways, such as apoptosis, in order to ensure an uncontrollable growth as well as resistance to various treatments. Dysregulation of the BCL-2 family of proteins that critically regulate the intrinsic apoptotic pathway contributes to the pathogenesis of cancer [1]. A major mechanism of cancer progression relies on the overexpression of anti-apoptotic BCL-2 proteins, such as BCL-2, BCL-XL and MCL-1, which steers cellular fate towards survival [1]. Anti-apoptotic members bind and neutralize the BH3 domains of multidomain pro-apoptotic BCL-2 members, BAX and BAK, and pro-apoptotic BH3-only proteins such as BIM and BID, thereby suppressing cell death [1]. Often, more resistant tumors to cancer therapeutics concomitantly suppress or degrade pro-apoptotic BH3-only proteins limiting the proteins available to directly activate BAX and BAK to promote apoptosis [2]. Inhibitors of anti-apoptotic BCL-2 proteins targeting specific members have been developed with promising clinical results; however, their use has been limited in cancers that rely on additional antiapoptotic mechanisms for survival [1, 2]. These findings suggest the need for alternative therapeutic strategies that have the potential to overcome such blockades of tumor cell death. Pro-apoptotic BAX is a critical member of the BCL-2 protein family. Following apoptotic stimuli, activating BH3-only proteins interact with the trigger site on BAX resulting in a series of structural changes that culminate in the translocation of BAX from the cytosol to the mitochondria outer membrane (MOM) [3, 4]. Once anchored to the MOM, BAX oligomerizes and creates a pore where apoptogenic factors, such as cytochrome c, escape and activate the caspase cascade that orchestrates apoptosis. In the vast majority of cancer cells, BAX is expressed in an inactive conformation or suppressed by anti-apoptotic proteins [1, 2]. Mutations in BAX that may cause its inactivation are present at low frequency in different tumors [5]. Therefore, we hypothesized that targeting direct activation of BAX to promote apoptosis may offer an alternative therapeutic strategy for cancer. To assess the therapeutic potential of direct BAX activation, we set out to discover small molecules that mimic the BAX-activating interactions of BIM with the N-terminal trigger site of BAX [6, 7]. We used a pharmacophore model and medicinal chemistry to rationally design BAX trigger site binders based on previous structural models of the BIM BH3 helix and BAM7 compound. Synthesized compounds were optimized based on a binding assay that evaluates competition of fluorescein-labeled BIM BH3 helix from the BAX trigger site. We identified BAX trigger site activator 1 (BTSA1), a lead compound with high affinity and selectivity for the BAX trigger site against other anti-apoptotic BCL-2 proteins [7]. Moreover, using biochemical BAX activation assays we demonstrated that binding of BTSA1 to BAX induced all the necessary conformational changes that lead to BAX translocation and oligomerization to promote MOM permeabilization (Figure 1). We investigated the cell-based capacity of BTSA1 to induce apoptosis in a panel of acute myeloid leukemia (AML) cell lines that overexpress various anti-apoptotic proteins. BTSA1 promptly and robustly decreased cellular viability in a dose and time-dependent manner. BAX translocation to the OMM, cytochrome c release and activation of effector caspases were significantly accomplished at 4 hrs (Figure 1). The pro-apoptotic effect of BTSA1 was correlated with the protein levels of BAX and not of the anti-apoptotic BCL-2 proteins. Cellular specificity was confirmed with a weaker analogue of BTSA1, a pharmacological inhibitor of BAX mitochondrial channel and a siRNA knockdown of BAX. A biotinylated form of BTSA1 was synthesized for Editorial

Abstract 2455: BAM38 is a potent and selective pro-apoptotic BAX activator molecule that restores apoptosis in cancer cells.

Targeting key apoptosis regulators to overcome the apoptotic resistance of cancer cells is a highly attractive therapeutic strategy. The BCL-2 protein family includes both pro- and anti-apoptotic proteins that form a complex interaction network and regulate the critical balance between cellular life and death. BAX is a pro-apoptotic BCL-2 member that, when activated, undergoes a structural transformation, which converts BAX from an inactive conformation into a lethal one, creating mitochondrial pores. Although inactivating mutations in BAX have been identified with a very small frequency in tumors, cancer cells commonly ensure survival by overexpression of anti-apoptotic BCL-2 members, a feature that contributes to tumorigenesis and chemoresistance. Therefore, the vast majority of cancer cells contain functional but suppressed BAX. Our discovery of the BAX trigger site and BAX activator molecule 7 (BAM7), which directly triggers BAX activation and selective BAX-mediated cell death, provides a new opportunity for rational drug design to target BAX and promote cell death in tumors. Using an in silico structural similarity approach and a competitive fluorescence polarization assay, we identified a novel small molecule activator of BAX, BAM38, that has ten-fold improved binding potency compared to BAM7 and retains selectivity over anti-apoptotic BCL-2 proteins. NMR and molecular docking analysis demonstrates that BAM38 engages the BAX trigger site using increased complementary interactions. Biochemical assays indicate that BAM38 has correspondingly improved potency in triggering BAX conformational activation and BAX-mediated membrane permeabilization. In a panel of acute myeloid leukemia (AML) cells that overexpress distinct combinations of anti-apoptotic BCL-2 family proteins, BAM38 overcomes apoptotic blockade through induction of BAX-mediated apoptosis. We observe dose-dependent AML cell death that coincides with BAX translocation, cytochrome c release and caspase 3/7 activation. BAM38 synergizes with the BCL-2/BCL-X L inhibitor ABT-737 in resistant AML cells, highlighting the therapeutic potential of combined direct pharmacologic activation of BAX with targeted inhibition of anti-apoptotic BCL-2 and BCL-X L . Thus, our data suggest a novel small molecule approach to restoring cancer cell death by directly targeting BAX, and provides a lead molecule for further development into a next generation cancer therapeutic. Citation Format: Denis E. Reyna, Loren D. Walensky, Evripidis Gavathiotis. BAM38 is a potent and selective pro-apoptotic BAX activator molecule that restores apoptosis in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2455. doi:10.1158/1538-7445.AM2013-2455 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2471: Chemical optimization of direct and selective molecular activators of pro-apoptotic BAX for cancer therapy.

BAX is a pro-apoptotic BCL-2 family member that lies dormant in the cytosol until triggered by cellular stress to translocate to the mitochondria and form toxic oligomeric pores. The majority of cancer cells retain wild-type BAX and instead neutralize the death pathway by overexpressing anti-apoptotic BCL-2 family proteins. Thus, direct activation of BAX represents a pharmacologic opportunity to lower the apoptotic threshold in the setting of chemoresistance. Using a Stabilized Alpha-Helix of BCL-2 domain (SAHB) modeled after the BIM BH3 helix, we previously identified the “trigger site” for direct BAX activation at the confluence of alpha-helices 1 and 6. In silico screening for small molecule modulators of BAX led to our identification of BAX activator molecule 7 (BAM7), which selectively engages the trigger site and promotes BAX-mediated cell death (Gavathiotis et al, Nat Chem Biol, 2012). Here, we applied BAM7 to a panel of acute lymphoblastic leukemia cells and observe dose-responsive killing in the micromolar range. To improve the potency of anti-ALL activity, we undertook a systematic medicinal chemistry-based iteration of BAM7’s molecular features. A series of analogs demonstrate improved BAX binding activity based on a FITC-BIM SAHB/BAX competitive fluorescence polarization assay. The most potent binders were advanced to testing in a cellular model of anti-apoptotic protein-dependent chemoresistance. Whereas select BAM7 derivatives exhibited a potency similar to or greater than the BCL-2/BCL-X L inhibitor ABT-737 in a BCL-X L -dependent ALL cell line, the compounds significantly outperformed ABT-737 in the isogenic MCL-1-dependent ALL cells. Preliminary studies revealed favorable pharmacokinetic profiles for lead BAM7 derivatives, including oral bioavailability and central nervous system penetration. These early encouraging structure-activity relationship and pharmacokinetic data on chemically-optimized BAM7 analogs suggest that direct and selective activation of BAX may be a viable strategy for therapeutic induction of apoptosis in the context of cancer chemoresistance. Citation Format: Joseph A. Bellairs, Denis Reyna, Divakaramenon Sethumadhavan, Madhavi Neelagiri, Alex Broadhead, Christopher Baccei, Brian Koss, Joseph T. Opferman, Evripidis Gavathiotis, Loren D. Walensky. Chemical optimization of direct and selective molecular activators of pro-apoptotic BAX for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2013-2471