Bcl-2 proteins and apoptosis: choose your partner.

The Bcl-2 family protein Bax is a key effector of apoptosis. Lovell et al. (2008) now describe the reconstitution and regulation in liposomes of tBid-mediated membrane penetration and oligomerization of Bax. These and other recent studies shed new light on the control of permeabilization of the mitochondrial outer membrane during apoptosis.

BAP31 interacts with Sec61 translocons and promotes retrotranslocation of CFTRDeltaF508 via the derlin-1 complex.

BAP31 is an endoplasmic reticulum protein-sorting factor that associates with newly synthesized integral membrane proteins and controls their fate (i.e., egress, retention, survival, or degradation). BAP31 is itself an integral membrane protein and a constituent of several large protein complexes. Here, we show that a part of the BAP31 population interacts with two components of the Sec61 preprotein translocon, Sec61beta and TRAM. BAP31 associates with the N terminus of one of its newly synthesized client proteins, the DeltaF508 mutant of CFTR, and promotes its retrotranslocation from the ER and degradation by the cytoplasmic 26S proteasome system. Depletion of BAP31 reduces the proteasomal degradation of DeltaF508 and permits a significant fraction of the surviving protein to reach the cell surface. Of note, BAP31 also associates physically and functionally with the Derlin-1 protein disclocation complex in the DeltaF508 degradation pathway. Thus, BAP31 operates at early steps to deliver newly synthesized CFTRDeltaF508 to its degradation pathway.

MAPL SUMOylation of Drp1 Stabilizes an ER/Mitochondrial Platform Required for Cell Death.

There has been evidence that mitochondrial fragmentation is required for apoptosis, but the molecular links between the machinery regulating dynamics and cell death have been controversial. Indeed, activated BAX and BAK can form functional channels in liposomes, bringing into question the contribution of mitochondrial dynamics in apoptosis. We now demonstrate that the activation of apoptosis triggers MAPL/MUL1-dependent SUMOylation of the fission GTPase Drp1, a process requisite for cytochrome c release. SUMOylated Drp1 functionally stabilizes ER/mitochondrial contact sites that act as hotspots for mitochondrial constriction, calcium flux, cristae remodeling, and cytochrome c release. The loss of MAPL does not alter the activation and assembly of BAX/BAK oligomers, indicating that MAPL is activated downstream of BAX/BAK. This work demonstrates how interorganellar contacts are dynamically regulated through active SUMOylation during apoptosis, creating a stabilized platform that signals cytochrome c release.

DZ-2384 has a superior preclinical profile to taxanes for the treatment of triple-negative breast cancer and is synergistic with anti-CTLA-4 immunotherapy.

Triple-negative breast cancer (TNBC) is typically aggressive, difficult to treat, and commonly metastasizes to the visceral organs and soft tissues, including the lungs and the brain. Taxanes represent the most effective and widely used therapeutic class in metastatic TNBC but possess limiting adverse effects that often result in a delay, reduction, or cessation of their use. DZ-2384 is a candidate microtubule-targeting agent with a distinct mechanism of action and strong activity in several preclinical cancer models, with reduced toxicities. DZ-2384 is highly effective in patient-derived taxane-sensitive and taxane-resistant xenograft models of TNBC at lower doses and over a wider range relative to paclitaxel. When comparing compound exposure at minimum effective doses relative to safe exposure levels, the therapeutic window for DZ-2384 is 14-32 compared with 2.0 and less than 2.8 for paclitaxel and docetaxel, respectively. DZ-2384 is effective at reducing brain metastatic lesions when used at maximum tolerated doses and is equivalent to paclitaxel. Drug distribution experiments indicate that DZ-2384 is taken up more efficiently by tumor tissue but at equivalent levels in the brain compared with paclitaxel. Selective DZ-2384 uptake by tumor tissue may in part account for its wider therapeutic window compared with taxanes. In view of the current clinical efforts to combine chemotherapy with immune checkpoint inhibitors, we demonstrate that DZ-2384 acts synergistically with anti-CTLA-4 immunotherapy in a syngeneic murine model. These results demonstrate that DZ-2384 has a superior pharmacologic profile over currently used taxanes and is a promising therapeutic agent for the treatment of metastatic TNBC.

Translation initiation factor eIF4F modifies the dexamethasone response in multiple myeloma.

Enhanced protein synthesis capacity is associated with increased tumor cell survival, proliferation, and resistance to chemotherapy. Cancers like multiple myeloma (MM), which display elevated activity in key translation regulatory nodes, such as the PI3K/mammalian target of rapamycin and MYC-eukaryotic initiation factor (eIF) 4E pathways, are predicted to be particularly sensitive to therapeutic strategies that target this process. To identify novel vulnerabilities in MM, we undertook a focused RNAi screen in which components of the translation apparatus were targeted. Our screen was designed to identify synthetic lethal relationships between translation factors or regulators and dexamethasone (DEX), a corticosteroid used as frontline therapy in this disease. We find that suppression of all three subunits of the eIF4F cap-binding complex synergizes with DEX in MM to induce cell death. Using a suite of small molecules that target various activities of eIF4F, we observed that cell survival and DEX resistance are attenuated upon eIF4F inhibition in MM cell lines and primary human samples. Levels of MYC and myeloid cell leukemia 1, two known eIF4F-responsive transcripts and key survival factors in MM, were reduced upon eIF4F inhibition, and their independent suppression also synergized with DEX. Inhibition of eIF4F in MM exerts pleotropic effects unraveling a unique therapeutic opportunity.

Obatoclax is a direct and potent antagonist of membrane-restricted Mcl-1 and is synthetic lethal with treatment that induces Bim.

BACKGROUND: Obatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell lethality. The insolubility of this compound, however, has precluded the use of many classical drug-target interaction assays for its study. Thus, a direct demonstration of the proposed mechanism of action, and preferences for individual Bcl-2 family members, remain to be established. METHODS: Employing modified proteins and lipids, we recapitulated the constitutive association and topology of mitochondrial outer membrane Mcl-1 and Bak in synthetic large unilamellar liposomes, and measured bakdependent bilayer permeability. Additionally, cellular and tumor models, dependent on Mcl-1 for survival, were employed. RESULTS: We show that regulation of bilayer permeabilization by the tBid - Mcl-1 - Bak axis closely resemblesthe tBid - Bcl-XL - Bax model. Obatoclax rapidly and completely partitioned into liposomal lipid but also rapidly exchanged between liposome particles. In this system, obatoclax was found to be a direct and potent antagonist of liposome-bound Mcl-1 but not of liposome-bound Bcl-XL, and did not directly influence Bak. A 2.5 molar excess of obatoclax relative to Mcl-1 overcame Mcl-1-mediated inhibition of tBid-Bak activation. Similar results were found for induction of Bak oligomers by Bim. Obatoclax exhibited potent lethality in a cellmodel dependent on Mcl-1 for viability but not in cells dependent on Bcl-XL. Molecular modeling predicts that the 3-methoxy moiety of obatoclax penetrates into the P2 pocket of the BH3 binding site of Mcl-1. A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1. Systemic treatment of mice bearing Tsc2(+) (/) (-) Em-myc lymphomas (whose cells depend on Mcl-1 for survival) with obatoclax conferred a survival advantage compared to vehicle alone (median 31 days vs 22 days, respectively; p=0.003). In an Akt-lymphoma mouse model, the anti-tumor effects of obatoclax synergized with doxorubicin. Finally, treatment of the multiple myeloma KMS11 cell model (dependent on Mcl-1 for survival) with dexamethasone induced Bim and Bim-dependent lethality. As predicted for an Mcl-1 antagonist, obatoclax and dexamethasone were synergistic in this model. CONCLUSIONS: Taken together, these findings indicate that obatoclax is a potent antagonist of membranerestricted Mcl-1. Obatoclax represents an attractive chemical series to generate second generation Mcl-1 inhibitors.

Programming cancer cells for high expression levels of Mcl1.

The Bcl2 pro-survival protein family has long been recognized for its important contributions to cancer. At elevated levels relative to pro-apoptotic effector members, the survival proteins prevent cancer cells from initiating apoptosis in the face of many intrinsic tumour-suppressing pathways and extrinsic therapeutic treatments aimed at controlling tumorigenesis. Recent studies, including genome-wide analyses, have begun to focus attention on a particularly enigmatic member of the family-myeloid cell leukaemia 1 (Mcl1). For reasons that are not clear, Mcl1 in cancer cells is turned over rapidly, eliminated primarily through the ubiquitin-proteasome pathway. Moreover, the mechanistic aspects of this constitutive membrane-associated protein have not been fully elucidated. As the pro-cancer activity of Mcl1 requires elevated expression levels of the protein, the cancer genome adapts to ensure either high levels of synthesis or evasion of degradation, or both. Here, we focus on the complex strategies at play and their therapeutic implications.

Tailored fragments of roseophilin selectively antagonize Mcl-1 in vitro.

We have discovered a fragment of the natural product roseophilin, a member of the prodiginine family, that antagonizes Mcl-1 functions in a liposome-based assay for mitochondrial membrane permeabilization. By tailoring this substance such that it can participate in salt bridging with the protein surface, we have prepared the first prodiginine inspired structure that shows direct, saturable binding to a recombinant Bcl-2 family member in vitro.

Bcl-2-associated autophagy regulator Naf-1 required for maintenance of skeletal muscle.

Nutrient-deprivation autophagy factor-1 (NAF-1) was identified as an endoplasmic reticulum (ER) BCL-2-interacting protein, which functions to mediate the ability of ER BCL-2 to antagonize Beclin 1-dependent autophagy and depress ER calcium stores. In humans, a point mutation in Naf-1 (synonyms: Cisd2, Eris, Miner1 and Noxp70) is responsible for the neurodegenerative disorder Wolfram Syndrome 2. Here, we describe the generation and characterization of the Naf-1 gene deletion in mice. Naf-1 null mice display discernable clinical signs of degeneration at 2-3 months of age, with early evidence of significant defects in the structure and performance of skeletal muscle. Skeletal muscles from Naf-1 knockout mice demonstrate a significant shift towards slow-twitch (type I) fibers and greater resistance to muscle fatigue. Force-generating capacity is dramatically reduced in Naf-1(-/-) muscle. Consistent with its role in ER BCL-2-mediated regulation of autophagy and calcium flux, these physiological deficiencies were accompanied by augmented autophagy and dysregulated calcium homeostasis. In contrast, this also included adaptive enlargement of mitochondria with extensive cristae structures. Thus, NAF-1, a BCL-2-associated autophagy regulator, is required for homeostatic maintenance of skeletal muscle. Our findings uncover a novel pathway that is required for normal muscle maintenance, which may ultimately provide a novel therapeutic target for treating certain muscle pathologies.

Antagonism of Beclin 1-dependent autophagy by BCL-2 at the endoplasmic reticulum requires NAF-1.

In addition to mitochondria, BCL-2 is located at the endoplasmic reticulum (ER) where it is a constituent of several distinct complexes. Here, we identify the BCL-2-interacting protein at the ER, nutrient-deprivation autophagy factor-1 (NAF-1)-a bitopic integral membrane protein whose defective expression underlies the aetiology of the neurodegenerative disorder Wolfram syndrome 2 (WFS2). NAF-1 contains a two iron-two sulphur coordinating domain within its cytosolic region, which is necessary, but not sufficient for interaction with BCL-2. NAF-1 is displaced from BCL-2 by the ER-restricted BH3-only protein BIK and contributes to regulation of BIK-initiated autophagy, but not BIK-dependent activation of caspases. Similar to BCL-2, NAF-1 is found in association with the inositol 1,4,5-triphosphate receptor and is required for BCL-2-mediated depression of ER Ca(2+) stores. During nutrient deprivation as a physiological stimulus of autophagy, BCL-2 is known to function through inhibition of the autophagy effector and tumour suppressor Beclin 1. NAF-1 is required in this pathway for BCL-2 at the ER to functionally antagonize Beclin 1-dependent autophagy. Thus, NAF-1 is a BCL-2-associated co-factor that targets BCL-2 for antagonism of the autophagy pathway at the ER.

Bcl2 at the endoplasmic reticulum protects against a Bax/Bak-independent paraptosis-like cell death pathway initiated via p20Bap31.

Bap31 is an integral ER membrane protein which functions as an escort factor in the sorting of newly synthesized membrane proteins within the endoplasmic reticulum (ER). During apoptosis signaling, Bap31 is subject to early cleavage by initiator caspase-8. The resulting p20Bap31 (p20) fragment has been shown to initiate proapoptotic ER-mitochondria Ca2+ transmission, and to exert dominant negative (DN) effects on ER protein trafficking. We now report that ectopic expression of p20 in E1A/DNp53-transformed baby mouse kidney epithelial cells initiates a non-apoptotic form of cell death with paraptosis-like morphology. This pathway was characterized by an early rise in ER Ca2+ stores and massive dilation of the ER/nuclear envelope, dependent on intact ER Ca2+ stores. Ablation of the Bax/Bak genes had no effect on these ER/nuclear envelope transformations, and delayed but did not prevent cell death. ER-restricted expression of Bcl2 in the absence of Bax/Bak, however, delayed both ER/nuclear envelope dilation and cell death. This prosurvival role of Bcl2 at the ER thus extended beyond inhibition of Bax/Bak, and correlated with its ability to lower ER Ca2+ stores. Furthermore, these results indicate that ER restricted Bcl2 is capable of antagonizing not only apoptosis, but also a non-apoptotic, Bax/Bak independent, paraptosis-like form of cell death.

Mitochondrion-dependent N-terminal processing of outer membrane Mcl-1 protein removes an essential Mule/Lasu1 protein-binding site.

Mcl-1, a pro-survival member of the Bcl-2 family located at the mitochondrial outer membrane, is subject to constitutive ubiquitylation by the Bcl-2 homology 3-only E3 ligase, Mule/Lasu1, resulting in rapid steady-state degradation via the proteasome. Insertion of newly synthesized Mcl-1 into the mitochondrial outer membrane is dependent on its C-terminal transmembrane segment, but once inserted, the N terminus of a portion of the Mcl-1 molecules can be subject to proteolytic processing. Remarkably, this processing requires an intact electrochemical potential across the inner membrane. Three lines of evidence directed at the endogenous protein, however, indicate that the resulting Mcl-1ΔN isoform resides in the outer membrane: (i) full-length Mcl-1 and Mcl-1ΔN resist extraction by alkali but are accessible to exogenous protease; (ii) almost the entire populations of Mcl-1 and Mcl-1ΔN are accessible to the membrane-impermeant Cys-reactive agent 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid; and (iii) Mcl-1 and Mcl-1ΔN exhibit equivalent chemical cross-linking to Bak in intact mitochondria, an Mcl-1 binding partner located in the outer membrane. In addition to the Mule Bcl-2 homology 3 domain, we show that interaction between Mcl-1 and Mule also requires the extreme N terminus of Mcl-1, which is lacking in Mcl-1ΔN. Thus, Mcl-1ΔN does not interact with Mule, exhibits reduced steady-state ubiquitylation, evades the hyper-rapid steady-state degradation that is observed for full-length Mcl-1 in response to treatments that limit global protein synthesis, and confers resistance to UV stress-induced cell death.

Molecular dynamics study of small molecule inhibitors of the Bcl-2 family.

We carried out docking and molecular dynamics simulations on ABT-737 and obatoclax, which are inhibitors of the Bcl-2 family of proteins. We modeled the binding mode of ABT-737 with Bcl-x(L) , Bcl-2, and Mcl-1 and examined their dynamical behavior. We found that the binding of the chlorobiphenyl end of ABT-737 was quite stable across all three proteins. However, the phenylpiperazine linker group was dramatically more mobile in Mcl-1 compared to either Bcl-x(L) or Bcl-2. The S-phenyl group at the p4 binding site was well-anchored in Bcl-x(L) and Bcl-2 but was somewhat more mobile in Mcl-1 although the phenyl ring itself on average stayed close to the p4 binding site in Mcl-1. This greater mobility is likely due to the greater openness of the p3 and p4 binding sites on Mcl-1. The calculated binding free energies were consistent with the much weaker binding affinity of ABT-737 for Mcl-1. Obatoclax was predicted to bind at the p1 and p2 binding sites of Mcl-1 and the binding mode was quite stable during the molecular dynamics simulation with Mcl-1 wrapping around the molecule. The modeled binding mode suggests that obatoclax is able to inhibit all three proteins because it makes use of the p1 and p2 binding sites alone, which is a fairly narrow groove in all three proteins unlike the p4 binding site, which is much broader in Mcl-1.

Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis.

Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.

Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

Unique biology of Mcl-1: therapeutic opportunities in cancer.

Accumulating evidence suggests that Mcl-1 plays a critical pro-survival role in the development and maintenance of both normal and malignant tissues. Regulation of Mcl-1 expression occurs at multiple levels, allowing for either the rapid induction or elimination of the protein in response to different cellular events. This suggests that Mcl-1 can play an early role in response to signals directing either cell survival or cell death. Deregulation of pathways regulating Mcl-1 that result in its over-expression likely contribute to a cell's inability to properly respond to death signals possibly leading to cell immortalization and tumorigenic conversion. Correspondingly, Mcl-1 has been shown to be up-regulated in numerous hematological and solid tumor malignancies. Moreover, this up-regulation appears to be a factor in the resistance of some cancer types to conventional cancer therapies. Mechanisms that abrogate the pro-survival function of Mcl-1 either by diminishing its levels or inactivating its functional BH3 groove have shown promise for the combinational treatment with existing cancer therapies and as single agents in certain malignancies. Here we review the various pathways that regulate Mcl-1 expression and describe agents that are currently under development to modulate Mcl-1 activity for therapeutic benefit in oncology.

Mitochondrial membrane dynamics, cristae remodelling and apoptosis.

Mitochondria form a highly dynamic reticular network in living cells, and undergo continuous fusion/fission events and changes in ultrastructural architecture. Although significant progress has been made in elucidating the molecular events underlying these processes, their relevance to normal cell function remains largely unexplored. Emerging evidence, however, suggests an important role for mitochondrial dynamics in cellular apoptosis. The mitochondria is at the core of the intrinsic apoptosis pathway, and provides a reservoir for protein factors that induce caspase activation and chromosome fragmentation. Additionally, mitochondria modulate Ca2+ homeostasis and are a source of various metabolites, including reactive oxygen species, that have the potential to function as second messengers in response to apoptotic stimuli. One of the mitochondrial factors required for activation of caspases in most intrinsic apoptotic pathways, cytochrome c, is largely sequestered within the intracristae compartment, and must migrate into the boundary intermembrane space in order to allow passage across the outer membrane to the cytosol. Recent evidence argues that inner mitochondrial membrane dynamics regulate this process. Here, we review the contribution of mitochondrial dynamics to the intrinsic apoptosis pathway, with emphasis on the inner membrane.

The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B.

In the past decade, traditional yeast two-hybrid techniques have identified a plethora of interactions among soluble proteins operating within diverse cellular pathways. The discovery of associations between membrane proteins by genetic approaches, on the other hand, is less well established due to technical limitations. Recently, a split-ubiquitin system was developed to overcome this barrier, but so far, this system has been limited to the analysis of known membrane protein interactions. Here, we constructed unique split-ubiquitin-linked cDNA libraries and provide details for implementing this system to screen for binding partners of a bait protein, in this case BAP31. BAP31 is a resident integral protein of the endoplasmic reticulum, where it operates as a chaperone or cargo receptor and regulator of apoptosis. Here we describe a novel human member of the protein tyrosine phosphatase-like B (PTPLB) family, an integral protein of the endoplasmic reticulum membrane with four membrane-spanning alpha helices, as a BAP31-interacting protein. PTPLB turns over rapidly through degradation by the proteasome system. Comparisons of mouse cells with a deletion of Bap31 or reconstituted with human BAP31 indicate that BAP31 is required to maintain PTPLB, consistent with a chaperone or quality control function for BAP31 in the endoplasmic reticulum membrane.

Novel NAPRT specific antibody identifies small cell lung cancer and neuronal cancers as promising clinical indications for a NAMPT inhibitor/niacin co-administration strategy.

Tumor cells are particularly dependent on NAD+ due to higher rates of metabolism, DNA synthesis and repair. Nicotinamide phosphoribosyltransferase inhibitors (NAMPTis) inhibit NAD+ biosynthesis and represent promising new anti-cancer agents. However, clinical efficacy has been limited by toxicities demonstrating the need for drug combinations to broaden the therapeutic index. One potential combination involves niacin/NAMPTi co-administration. Niacin can rescue NAD+ biosynthesis through a parallel pathway that depends on nicotinic acid phosphoribosyltransferase (NAPRT) expression. Most normal tissues express NAPRT while a significant proportion of malignant cells do not, providing a possible selection marker for patients to achieve NAMPTi efficacy while minimizing toxicities. Here we identify and validate a novel highly NAPRT-specific monoclonal antibody (3C6D2) that detects functional NAPRT in paraffin embedded tissue sections by immunohistochemistry (IHC). NAPRT detection by 3C6D2 coincides with the ability of niacin to rescue cells from NAMPTi induced cytotoxicity in cell lines and animal xenograft models. 3C6D2 binds to an epitope that is unique to NAPRT among phosphoribosyltransferases. In a series of primary tumor samples from lung and brain cancer patients, we demonstrate that >70 % of human small cell lung carcinomas, glioblastomas and oligodendrogliomas lack NAPRT identifying them as potentially suitable indications for the NAMPT/niacin combination.