Obatoclax is a direct and potent antagonist of membrane-restricted Mcl-1 and is synthetic lethal with treatment that induces Bim. Nguyen, M; Cencic, R; Ertel, F; Bernier, C; Pelletier, J; Roulston, A; Silvius, JR; Shore, GC BMC cancer
15
568
2015
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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.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.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.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. | | | 26231047
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p73 engages A2B receptor signalling to prime cancer cells to chemotherapy-induced death. Long, JS; Schoonen, PM; Graczyk, D; O'Prey, J; Ryan, KM Oncogene
34
5152-62
2015
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Tumour cells often acquire the ability to escape cell death, a key event leading to the development of cancer. In almost half of all human cancers, the capability to induce cell death is reduced by the mutation and inactivation of p53, a tumour suppressor protein that is a central regulator of apoptosis. As a result, there is a crucial need to identify different cell death pathways that could be targeted in malignancies lacking p53. p73, the closely related p53 family member, can regulate many p53 target genes and therefore some of the same cellular responses as p53. Unlike p53, however, p73 is seldom mutated in cancer, making it an attractive, alternative death effector to target. We report here the ability of p73 to upregulate the expression of the A2B receptor, a recently characterized p53 target that effectively promotes cell death in response to extracellular adenosine--a metabolite that accumulates during various forms of cellular stress. Importantly, we show that p73-dependent stimulation of A2B signalling markedly enhances apoptosis in cancer cells that are devoid of p53. This mode of death is caspase- and puma-dependent, and can be prevented by the overexpression of anti-apoptotic Bcl-X(L). Moreover, treatment of p53-null cancer cells with the chemotherapeutic drug adriamycin (doxorubicin) induces A2B in a p73-dependent manner and, in combination with an A2B agonist, substantially enhances apoptotic death. We therefore propose an alternate and distinct p53-independent pathway to stimulate programmed cell death involving p73-mediated engagement of adenosine signalling. | | | 25659586
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Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells. Papaianni, E; El Maadidi, S; Schejtman, A; Neumann, S; Maurer, U; Marino-Merlo, F; Mastino, A; Borner, C PloS one
10
e0126645
2015
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Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells. | | | 26030884
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Biomarkers in advanced larynx cancer. Bradford, CR; Kumar, B; Bellile, E; Lee, J; Taylor, J; D'Silva, N; Cordell, K; Kleer, C; Kupfer, R; Kumar, P; Urba, S; Worden, F; Eisbruch, A; Wolf, GT; Teknos, TN; Prince, ME; Chepeha, DB; Hogikyan, ND; Moyer, JS; Carey, TE The Laryngoscope
124
179-87
2014
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To determine if tumor biomarkers were predictive of outcome in a prospective cohort of patients with advanced larynx cancer treated in a phase II clinical trial.Prospectively collected biopsy specimens from 58 patients entered into a Phase II trial of organ preservation in advanced laryngeal cancer were evaluated for expression of a large panel of biomarkers, and correlations with outcome were determined.Tissue microarrays were constructed from pretreatment biopsies and stained for cyclin D1, CD24, EGFR, MDM2, PCNA, p53, survivin, Bcl-xL, Bcl-2, BAK, rhoC, and NFκB. Pattern of invasion and p53 mutations were assessed. Correlations with overall survival (OS), disease-specific survival (DSS), time free from indication of surgery, induction chemotherapy response, and chemoradiation response were determined. Cox models were used to assess combinations of these biomarkers.Low expression of BAK was associated with response to induction chemotherapy. Low expression of BAK and cytoplasmic NFκB was associated with chemoradiation response. Aggressive histologic growth pattern was associated with response induction chemotherapy. Expression of cyclin D1 was predictive of overall and disease-specific survival. Overexpression of EGFR was also associated with an increased risk of death from disease. Bcl-xL expression increased significantly in persistent/recurrent tumors specimens when compared to pretreatment specimens derived from the same patient (P = 0.0003).Evaluation of biomarker expression in pretreatment biopsy specimens can lend important predictive and prognostic information for patients with advanced larynx cancer. | Immunohistochemistry | | 23775802
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BM-1197: a novel and specific Bcl-2/Bcl-xL inhibitor inducing complete and long-lasting tumor regression in vivo. Bai, L; Chen, J; McEachern, D; Liu, L; Zhou, H; Aguilar, A; Wang, S PloS one
9
e99404
2014
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Bcl-2 and Bcl-xL are critical regulators of apoptosis that are overexpressed in a variety of human cancers and pharmacological inhibition of Bcl-2 and Bcl-xL represents a promising strategy for cancer treatment. Using a structure-based design approach, we have designed BM-1197 as a potent and efficacious dual inhibitor of Bcl-2 and Bcl-xL. BM-1197 binds to Bcl-2 and Bcl-xL proteins with Ki values less than 1 nM and shows greater than 1,000-fold selectivity over Mcl-1. Mechanistic studies performed in the Mcl-1 knockout mouse embryonic fibroblast (MEF) cells revealed that BM-1197 potently disassociates the heterodimeric interactions between anti-apoptotic and pro-apoptotic Bcl-2 family proteins, concomitant with conformational changes in Bax protein, loss of mitochondrial membrane potential and subsequent cytochrome c release to the cytosol, leading to activation of the caspase cascade and apoptosis. BM-1197 exerts potent growth-inhibitory activity in 7 of 12 small cell lung cancer cell lines tested and induces mechanism-based apoptotic cell death. When intravenously administered at daily or weekly in H146 and H1963 small-cell lung cancer xenograft models, it achieves complete and long-term tumor regression. Consistent with its targeting of Bcl-xL, BM-1197 causes transit platelet reduction in mice. Collectively, our data indicate that BM-1197 is a promising dual Bcl-2/Bcl-xL inhibitor which warrants further investigation as a new anticancer drug. | | | 24901320
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Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer. Nakajima, W; Hicks, MA; Tanaka, N; Krystal, GW; Harada, H Cell death & disease
5
e1052
2014
Afficher le résumé
The sensitivity to ABT-737, a prototype BH3 mimetic drug, varies in a broad range in small cell lung cancer (SCLC) cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents. | | | 24525728
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Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival. Eichhorn, JM; Alford, SE; Sakurikar, N; Chambers, TC Experimental cell research
322
415-24
2014
Afficher le résumé
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. | | | 24556425
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Downregulation of miR-23a and miR-27a following experimental traumatic brain injury induces neuronal cell death through activation of proapoptotic Bcl-2 proteins. Sabirzhanov, B; Zhao, Z; Stoica, BA; Loane, DJ; Wu, J; Borroto, C; Dorsey, SG; Faden, AI The Journal of neuroscience : the official journal of the Society for Neuroscience
34
10055-71
2014
Afficher le résumé
MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level. To identify miRs that may regulate neuronal cell death after experimental traumatic brain injury (TBI), we profiled miR expression changes during the first several days after controlled cortical impact (CCI) in mice. miR-23a and miR-27a were rapidly downregulated in the injured cortex in the first hour after TBI. These changes coincided with increased expression of the proapoptotic Bcl-2 family members Noxa, Puma, and Bax. In an etoposide-induced in vitro model of apoptosis in primary cortical neurons, miR-23a and miR-27a were markedly downregulated as early as 1 h after exposure, before the upregulation of proapoptotic Bcl-2 family molecules. Administration of miR-23a and miR-27a mimics attenuated etoposide-induced changes in Noxa, Puma, and Bax, reduced downstream markers of caspase-dependent (cytochrome c release and caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways, and limited neuronal cell death. In contrast, miRs hairpin inhibitors enhanced etoposide-induced neuronal apoptosis and caspase activation. Importantly, administration of miR-23a and miR-27a mimics significantly reduced activation of Puma, Noxa, and Bax as well as attenuated markers of caspase-dependent and -independent apoptosis after TBI. Furthermore, miR-23a and miR-27a mimics significantly attenuated cortical lesion volume and neuronal cell loss in the hippocampus after TBI. These findings indicate that post-traumatic decreases in miR-23a and miR-27a contribute to neuronal cell death after TBI by upregulating proapoptotic Bcl-2 family members, thus providing a novel therapeutic target. | | | 25057207
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The Bcl-2 protein family member Bok binds to the coupling domain of inositol 1,4,5-trisphosphate receptors and protects them from proteolytic cleavage. Schulman, JJ; Wright, FA; Kaufmann, T; Wojcikiewicz, RJ The Journal of biological chemistry
288
25340-9
2013
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Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895-1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis. | | | 23884412
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Paclitaxel-induced apoptosis is BAK-dependent, but BAX and BIM-independent in breast tumor. Miller, AV; Hicks, MA; Nakajima, W; Richardson, AC; Windle, JJ; Harada, H PloS one
8
e60685
2013
Afficher le résumé
Paclitaxel (Taxol)-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death) plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim(-/-) MEFs (mouse embryonic fibroblasts), the bim(-/-) mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak (-/-) MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax(-/-) MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance. | | | 23577147
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