Function Of Bcl-2 Family Proteins Research Articles

Co-operation of MCL-1 and BCL-XL anti-apoptotic proteins in stromal protection of MM cells from carfilzomib mediated cytotoxicity.

BCL-2 family proteins are important for tumour cell survival and drug resistance in multiple myeloma (MM). Although proteasome inhibitors are effective anti-myeloma drugs, some patients are resistant and almost all eventually relapse. We examined the function of BCL-2 family proteins in stromal-mediated resistance to carfilzomib-induced cytotoxicity in MM cells. Co-cultures employing HS5 stromal cells were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1-hour pulse method. The expression of BCL-2 family proteins was assessed by flow cytometry and WB. Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB. CFZ induced dose-dependent cell death of MM cells, primarily mediated by apoptosis. Culture of MM cells on HS-5 stromal cells resulted in reduced cytotoxicity to CFZ in a cell contact-dependent manner, upregulated expression of MCL-1 and increased dependency on BCL-XL. Inhibiting BCL-XL or MCL-1 with BH-3 mimetics abrogated stromal-mediated protection only at high doses, which may not be achievable in vivo. However, combining BH-3 mimetics at sub-therapeutic doses, which alone were without effect, significantly enhanced CFZ-mediated cytotoxicity even in the presence of stroma. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins. Stromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.

Phenome-Wide Scan Identifies Hematologic Traits Associated with Genetically-Determined Expression of BCL-2 Family Genes

BCL2 family proteins represent attractive and druggable targets for hematologic malignancy. Pro-survival proteins BCL2, BCL-XL, and MCL1 are key targets of BH3 mimetic and inhibitory drugs that are in clinical trials and/or clinical use for hematologic malignancies. Although the mechanism by which BH3-mimetic drugs bind anti-apoptotic family members is well-understood, less is known about off-target impacts of these drugs across the phenome. Furthermore, the impact of other BCL2 family proteins might have on clinically relevant phenotypes has not been explored. The development of and implementation of drugs that modulate the function of BCL2 family proteins could be enhanced by understanding gene-specific impacts on human phenotypes. Understanding the phenomic impacts of modulating BCL2 family expression has potential to enhance efficient development and inform clinical investigation of BCL2 modulating therapeutics for both hematologic and non-hematologic malignancy by identifying novel phenotype associations as well as by identifying potential side effects. In this study, we leveraged population-level differences in genetically determined gene expression of eighteen BCL-2 family genes to identify phenotypes that associate with altered genetically determined expression. We performed transcriptome-wide association studies (TWAS) utilizing GWAS summary statistics for 357 unique ICD-10 CM diagnosis codes and 31 unique hematologic traits from the UK Biobank V3 release (UKBB v3; n ~500,000). Summary statistics-based TWAS was applied using tissue-specific weights generated by Joint Tissue Integration (JTI) modeling of all 49 GTEx v8 tissues (Zhou et al., Nature Genetics 2020). From there, significant associations with eighteen core BCL2-family genes were pulled for analysis. This approach identified 25 unique phenotypes associated with expression of BCL2 family genes, given a Bonferroni-adjusted significance cutoff of p=1.5e-07. Gene-phenotype associations (Table 1) were remarkably enriched for hematologic phenotypes derived from complete blood count data, and demonstrated considerable overlap between cell death genes, with only three of these phenotypes uniquely mapping to a single gene. Conversely, BCL2 family genes were significantly enriched in hematologic trait datasets (p<0.01). BCL2L11, encoding BIM, maps to the most unique phenotypes (n=15), while BCL2 and BAX, which are highly conserved, have only four and three unique phenotype associations respectively. BBC3, PMAIP1, and BNIP3 did not have significant associations, suggesting that modulating genetically-determined expression levels of these genes alone contributes modestly at most to changes in individual human phenotypes. BCL2L1, which encodes BCL-X, exhibits associations with bi-directional effect sizes across different tissues (Figure 1). This is consistent with the known divergent biological functions of long versus short isoforms of this gene product (BCL-xL and BCL-xS). Though BCL-2 family members are thought to have redundant functions across members within the same subclassification, (e.g. BH3-only, pro-survival, effector), not all genes within these subclasses consistently exhibited the same phenotype associations. More strikingly, genes within the same class that shared phenotype associations did not always exhibit the same directionality of effect, suggesting that these genes may have divergent roles contributing to these phenotypes. Overall, our findings support the premise that BCL2-family gene expression distinctively targets hematologic phenotypes. The results also suggest that genes with canonically "conserved” functions may have biologically unique contributions to hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Glimpse of Programmed Cell Death Among Bacteria, Animals, and Plants.

Programmed cell death (PCD) in animals mainly refers to lytic and non-lytic forms. Disruption and integrity of the plasma membrane are considered as hallmarks of lytic and apoptotic cell death, respectively. These lytic cell death programs can prevent the hosts from microbial pathogens. The key to our understanding of these cases is pattern recognition receptors, such as TLRs in animals and LRR-RLKs in plants, and nod-like receptors (NLRs). Herein, we emphatically discuss the biochemical and structural studies that have clarified the anti-apoptotic and pro-apoptotic functions of Bcl-2 family proteins during intrinsic apoptosis and how caspase-8 among apoptosis, necroptosis, and pyroptosis sets the switchable threshold and integrates innate immune signaling, and that have compared the similarity and distinctness of the apoptosome, necroptosome, and inflammasome. We recapitulate that the necroptotic MLKL pore, pyroptotic gasdermin pore, HR-inducing resistosome, and mitochondrial Bcl-2 family all can form ion channels, which all directly boost membrane disruption. Comparing the conservation and unique aspects of PCD including ferrroptosis among bacteria, animals, and plants, the commonly shared immune domains including TIR-like, gasdermin-like, caspase-like, and MLKL/CC-like domains act as arsenal modules to restructure the diverse architecture to commit PCD suicide upon stresses/stimuli for host community.

Co-Operation of MCL-1 and BCL-X L Anti-Apoptotic Proteins in Stromal Protection of MM Cells from Carfilzomib Mediated Cytotoxicity

Introduction: Expression and function of BCL-2 family proteins in multiple myeloma (MM) is important for tumour cell survival and drug resistance, and may partly mediate the protective effect of bone marrow (BM) stroma. Proteasome inhibitors (PIs) are a major class of anti-myeloma drugs, however some patients are resistant and almost all eventually relapse. Carfilzomib is a second generation, irreversible proteasome inhibitor, which is effective in both newly diagnosed and relapsed/refractory disease, but drug resistance and relapse occur.Aim: We aimed to study the role of BCL-2 family proteins in Carfilzomib (CFZ) mediated cytotoxicity in the presence of stroma, focusing on priming of the pro-apoptotic protein, BIM.Methods: A panel of Human Myeloma Cell Lines (HMCL) and primary MM cells were exposed to Carfilzomib, and cell death assessed using Annexin V/PI staining and flow cytometry. Co-cultures employing the HS5 stromal cell line were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1hr pulse, to recapitulate the pharmacokinetics of drug exposure in patients. The expression of BCL-2 family proteins was assessed by flow cytometry and Western Blot (WB). The compounds S63845, ABT-199 (Venetoclax) and A-1331852 were used to inhibit MCL-1, BCL-2 and BCL-X L pro-survival proteins respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB.Results: CFZ induced dose and time-dependent cell death of HMCL and primary MM cells. This was primarily mediated by apoptosis as shown by AnV/PI staining (eg. MM1.s at 25nM CFZ: 34±6% apoptosis at 24h, 47±9% at 48h, p=0.016; 50nM CFZ: 60 ±3.8% at 24h vs 79±6% at 48h, p=0.004, mean ±SEM, n=3), and by reduced cytotoxicity in MM1.s BAX/BAK KO cells (100nM CFZ: 10±1% cell death vs 42±5% in WT at 6h, 33±14% vs 99±0.5% at 24h; p=0.0005, n=3). CFZ cytotoxicity was significantly reduced when MM1.s cells were co-cultured with HS-5 (50nM CFZ: 53±4% apoptosis vs 79±4% in suspension, at 48h, p=0.02, n=3) confirming the protective effect of stromal cells. Similar results were seen with H929, KMS27 and KMS12BM HMCL (Figure 1). Co-culture of MM1.s with HS-5 cells induced upregulation of MCL-1 and BCL-X L and downregulation of BCL-2. Combined inhibition of BCL-X L and MCL-1 at concentrations where single agents had minimal effect, resulted ina marked increase in apoptosis on stroma (Ctrl: 9±1%, S63845 100nM:12±1.4%, A-1331852 1nM: 17±3%, S63845+A-1331852: 60±9%; 48h, p=0.0001, n=3) (Figure 3). BIM immunoprecipitation showed that in the presence of the MCL-1 inhibitor, S63845, there was increased BCL-X L binding to BIM; conversely when BCL-X L was inhibited by A-1331852, an increase in MCL-1/BIM complexes was seen. Exposure of MM cells to CFZ in stromal co-cultures led to a further increase in the expression of BCL-X L and MCL-1. Because BCL-X L and MCL-1 were upregulated both by stromal interaction and by CFZ (Figure 2), we tested the cooperativity of these anti-apoptotic proteins on resistance to CFZ. CFZ-induced cytotoxicity was significantly enhanced by the addition of single BH3 mimetics in the presence of HS-5: cell death 48h, BCL-X L A-133182 5nM: 40±12%, CFZ 25nM: 57±10%, CFZ+A-1331852: 94±2%, p=0.0065, n=3. Cell death with MCL-1 inhibitor S63845 200nM: 12±3%, CFZ 25nM:40±13%, S63845+CFZ: 54±18%, p=0.02, n=3). However, combined BCL-X L and MCL-1 inhibition, using lower concentrations of BH3 mimetics, potently sensitized MM cells to CFZ-mediated cytotoxicity in the presence of stroma (% killing at CFZ 25nM + A-1331852 1nM: 48±8%; CFZ+S63845 100nM: 38±5%; CFZ+A-1331852+S63845: 92±2%, p<0.0001, n=3, Figure 3).Conclusion: BCL-X L and MCL-1 are upregulated in MM cells by stromal interaction, and also in response to CFZ. The upregulation of pro-survival proteins by stroma may mediate MM cell resistance to PIs, and blockade of both BCL-X L and MCL-1 is required in order to abrogate the protective effect of stroma and restore sensitivity to CFZ. Combination of BH-3 mimetic drugs at subtherapeutic doses with proteasome inhibitors may be a potential therapeutic strategy. [Display omitted] DisclosuresHuang: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Khwaja: Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yong: Janssen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding.

Discovery of Novel PTP1B Inhibitors Derived from the BH3 Domain of Proapoptotic Bcl-2 Proteins with Antidiabetic Potency.

BH3 peptide analogues are generally believed to exhibit great potency as cancer therapeutics via targeting antiapoptotic Bcl-2 proteins. Here, we describe the synthesis and identification of a new class of palmitoylated peptide BH3 analogues derived from the core region (h1-h4) of BH3 domains of proapoptotic Bcl-2 proteins and as alternative PTP1B inhibitors with antidiabetic potency in vitro and in vivo. PTP1B inhibitors are attractive for treatment of type 2 diabetes. We design the analogues using a simple lipidation approach and discovered novel lead analogues with promising antidiabetic potency in vitro and in vivo. The results presented here expanded the alternative target and function for the BH3 peptide analogues from one member Bim to other members of the proapoptotic Bcl-2 proteins and emphasize their therapeutic potential in T2DM. Furthermore, our findings may provide new proof of the regulatory function of Bcl-2 family proteins in mitochondrial nutrient and energy metabolism.

Apoptotic stress induces Bax-dependent, caspase-independent redistribution of LINC complex nesprins

The canonical function of Bcl-2 family proteins is to regulate mitochondrial membrane integrity. In response to apoptotic signals the multi-domain pro-apoptotic proteins Bax and Bak are activated and perforate the mitochondrial outer membrane by a mechanism which is inhibited by their interaction with pro-survival members of the family. However, other studies have shown that Bax and Bak may have additional, non-canonical functions, which include stress-induced nuclear envelope rupture and discharge of nuclear proteins into the cytosol. We show here that the apoptotic stimuli cisplatin and staurosporine induce a Bax/Bak-dependent degradation and subcellular redistribution of nesprin-1 and nesprin-2 but not nesprin-3, of the linker of nucleoskeleton and cytoskeleton (LINC) complex. The degradation and redistribution were caspase-independent and did not occur in Bax/Bak double knockout (DKO) mouse embryo fibroblasts (MEFs). Re-expression of Bax in Bax/Bak DKO MEFs restored stress-induced redistribution of nesprin-2 by a mechanism which requires Bax membrane localization and integrity of the α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. We found that nesprin-2 interacts with Bax in close proximity to perinuclear mitochondria in mouse and human cells. This interaction requires the mitochondrial targeting and N-terminal region but not the BH3 domain of Bax. Our results identify nesprin-2 as a Bax binding partner and also a new function of Bax in impairing the integrity of the LINC complex.

Bcl-2 family proteins, beyond the veil.

Apoptosis is an important part of both health and disease and is often regulated by the BCL-2 family of proteins. These proteins are either pro- or anti-apoptotic, existing in a delicate balance during homeostasis. They are best known for their role in regulating the activation of caspases and the execution of a cell in response to a variety of stimuli. However, it is often forgotten that these BCL-2 family proteins also have important roles to play in cell maintenance that are not associated with apoptosis. These include roles in regulating processes such as cell cycle progression, mitochondrial function, autophagy, intracellular calcium concentration, glucose and lipid metabolism, and the unfolded protein response. In addition to these established alternate functions, further discoveries are being made that have potential therapeutic benefits in diseases such as cancer. BOK, a BCL-2 family protein thought comparable to multidomain pro-apoptotic proteins BAX and BAK, has recently been identified as a key player in metabolism of and resistance to the commonly used chemotherapeutic 5-FU. As a result of such findings, which could see the potential use of BOK as a biomarker for 5-FU sensitivity or mimetic molecules as a resensitization strategy, new targets and mechanisms of pathology may arise from further investigation into the realm of alternate functions of BCL-2 family proteins.

Non-apoptotic functions of BCL-2 family proteins.

The BCL-2 family proteins are major regulators of the apoptosis process, but the mechanisms by which they regulate this process are only partially understood. It is now well documented that these proteins play additional non-apoptotic roles that are likely to be related to their apoptotic roles and to provide important clues to cracking their mechanisms of action. It seems that these non-apoptotic roles are largely related to the activation of cellular survival pathways designated to maintain or regain cellular survival, but, if unsuccessful, will switch over into a pro-apoptotic mode. These non-apoptotic roles span a wide range of processes that include the regulation of mitochondrial physiology (metabolism, electron transport chain, morphology, permeability transition), endoplasmic reticulum physiology (calcium homeostasis, unfolded protein response (UPR)), nuclear processes (cell cycle, DNA damage response (DDR)), whole-cell metabolism (glucose and lipid), and autophagy. Here we review all these different non-apoptotic roles, make an attempt to link them to the apoptotic roles, and present many open questions for future research directions in this fascinating field.

A Review on Structures and Functions of Bcl-2 Family Proteins from Homo sapiens.

Cancer cells evade apoptosis, which is regulated by proteins of Bcl-2 family in the intrinsic pathways. Numerous experimental three-dimensional (3D) structures of the apoptotic proteins and the proteins bound with small chemical molecules/peptides/proteins have been reported in the literature. In this review article, the 3D structures of the Bcl-2 family proteins from Homo sapiens and as well complex structures of the anti-apoptotic proteins bound with small molecular inhibitors reported in the literature to date have been comprehensively listed out and described in detail. Moreover, the molecular mechanisms by which the Bcl-2 family proteins modulate the apoptotic processes and strategies for designing antagonists to anti-apoptotic proteins have been concisely discussed.

Bcl-2 family proteins: master regulators of cell survival.

The most prominent function of proteins of the Bcl-2 family is regulation of the initiation of intrinsic (mitochondrial) pathways of apoptosis. However, recent research has revealed that in addition to regulation of mitochondrial apoptosis, proteins of the Bcl-2 family play important roles in regulating other cellular pathways with a strong impact on cell survival like autophagy, endoplasmic reticulum (ER) stress response, intracellular calcium dynamics, cell cycle progression, mitochondrial dynamics and energy metabolism. This review summarizes the recent knowledge about functions of Bcl-2 family proteins that are related to cell survival.

BH3 Profiling Discriminates Response to Cytarabine-Based Treatment of Acute Myelogenous Leukemia

As acute myelogenous leukemia (AML) patient response to cytarabine-based standard-of-care treatment is variable, stratification into subgroups by biomarker-predicted response may lead to improved clinical outcomes. Here, we assess cell mitochondrial depolarization to proapoptotic signaling BH3-only peptides as a surrogate for the function of Bcl-2 family proteins to address clinical response to cytarabine-based therapy in patients with AML (N = 62). Peripheral blood mononuclear cell (PBMC) or bone marrow aspirate specimens were obtained from newly diagnosed patients with AML, viably preserved, and assayed by flow cytometry following BH3 profile assay with individual BH3 peptides. Mann-Whitney analysis indicates biomarker correlation with response to induction therapy: Notably, BIM priming was highly significant (P = 2 × 10(-6)) with a compelling sensitivity/specificity profile [area under curve (AUC) = 0.83; 95% confidence interval (CI), 0.73-0.94; P = 2 × 10(-10)]. Multivariate analysis indicates improved profiles for BIM readout + patient age (AUC = 0.89; 95% CI, 0.81-0.97) and BIM + patient age + cytogenetic status (AUC = 0.91; 95% CI, 0.83-0.98). When patients were stratified by cytogenetic status, BIM readout was significant for both intermediate (P = 0.0017; AUC = 0.88; 95% CI, 0.71-1.04) and unfavorable (P = 0.023; AUC = 0.79; 95% CI, 0.58-1.00) risk groups, demonstrating predictive power independent of cytogenetics. Additional analyses of secondary clinical endpoints displayed correlation between overall survival (P = 0.037) and event-free survival (P = 0.044) when patients were stratified into tertiles by BIM peptide response. Taken together, these results highlight the potential utility of BH3 profiling in personalized diagnostics of AML by offering actionable information for patient management decisions.

Mcl-1 Dependence Predicts Response To Vorinostat and Gemtuzumab Ozogamicin In Acute Myeloid Leukemia

IntroductionThe outcome of older adults with acute myeloid leukemia (AML) using standard curative-intent chemotherapeutics remains dismal because of increasing risks of treatment-related mortality and therapeutic resistance associated with advancing patient age, accumulation of medical comorbidities, and changing disease biology. As a result, current expert guidelines, e.g. by the National Comprehensive Cancer Network (NCCN), recommend that these patients should receive investigational therapies. Still, investigational therapeutics typically exhibit response in only a subset of patients, and factors such as age or cytogenetics are often insufficient to predict treatment success with high accuracy. Here, we utilized BH3 profiling, a method for assessing mitochondrial functionality in apoptosis signaling, as a biomarker strategy for identification of patients likely to exhibit clinical response to an investigational regimen of a histone deacetylase (HDAC) inhibitor, vorinostat, in combination with gemtuzumab ozogamicin (GO), an immunoconjugate consisting of an anti-CD33 antibody and a toxic calicheamicin moiety. Cytotoxic effects of these agents have been shown to involve mitochondrial pathways of apoptosis, providing the scientific rationale for our study. Patients and MethodsThirty-one adults aged 60 years or older received treatment with vorinostat and GO for newly diagnosed AML (NCT00673153). For 26 of these patients, pretreatment peripheral blood mononuclear cell (PBMC) or bone marrow (BM) aspirate specimens were available. Blinded to outcomes, we analyzed these specimens by flow cytometry-based BH3 profiling. This assay indirectly measures induction of mitochondrial outer membrane permeabilization in response to treatment with BH3-only peptides, including activators (e.g. BIM) and sensitizers (e.g. NOXA, PUMA, BAD, HRK, BMF) as a surrogate for the function of Bcl-2 family proteins. Findings from these profiling studies were then correlated with disease characteristics and treatment outcome. ResultsBH3 profiling was technically successful in 23 of 26 tested specimens. Log regression analyses indicated a correlation between priming with NOXA, a selective modulator of the anti-apoptotic protein Mcl-1, and response to induction therapy (i.e. achievement of complete remission [CR]/CRp vs. resistance; p=.027). Using the area under the receiver operating curve (AUC) to quantify the accuracy of outcome prediction, the % priming achieved with NOXA yielded an AUC of 0.83 (95% CI: 0.65-1.00; p=.00042). After adjustment for age, the AUC associated with NOXA priming increased further (AUC=0.88 [0.75-1.00]). The % priming with NOXA was also statistically significantly associated with the duration of overall survival (OS; p=.027), with an AUC of 0.87 (0.64-1.00; p=.0017). Patients in the highest tertile of NOXA priming exhibited a median OS of 453.5 days vs. the other patients median OS of 132 days (p=.12 Mantel-Cox). ConclusionOur data show that the degree of priming indicated by NOXA identifies Mcl-1 dependence that is associated with response and outcome of vorinostat/GO in older adults with AML. This finding would be compatible with a pivotal role of Bcl-2 family protein-mediated mitochondrial depolarization for the clinical efficacy of GO and vorinostat, consistent with previous in vitro studies. Importantly, Mcl-1 -dependence as indicated by BH3 profiling of NOXA response may predict response to investigational therapeutic regimens for AML and, if integrated as predictive biomarker, may improve patient outcomes through identifying subsets of patients likely to respond to treatment with HDAC inhibitors and immunoconjugates including calicheamicins when given in combination. Disclosures:Pierceall:Eutropics: Employment, Equity Ownership. Lena:Eutropics: Employment, Equity Ownership. Blake:Noel Blake: Employment, Equity Ownership. Elaschoff:Eutropics: Consultancy. Cardone:Eutropics: Employment, Equity Ownership.

Multiple Functions of BCL-2 Family Proteins

BCL-2 family proteins are the regulators of apoptosis, but also have other functions. This family of interacting partners includes inhibitors and inducers of cell death. Together they regulate and mediate the process by which mitochondria contribute to cell death known as the intrinsic apoptosis pathway. This pathway is required for normal embryonic development and for preventing cancer. However, before apoptosis is induced, BCL-2 proteins have critical roles in normal cell physiology related to neuronal activity, autophagy, calcium handling, mitochondrial dynamics and energetics, and other processes of normal healthy cells. The relative importance of these physiological functions compared to their apoptosis functions in overall organismal physiology is difficult to decipher. Apoptotic and noncanonical functions of these proteins may be intertwined to link cell growth to cell death. Disentanglement of these functions may require delineation of biochemical activities inherent to the characteristic three-dimensional shape shared by distantly related viral and cellular BCL-2 family members.

Ethyl 2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017): A Novel Scaffold That Resensitizes Multidrug Resistant Leukemia Cells to Chemotherapy

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer, and there is a pressing need for new therapies. We have recently developed ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017), derived from a dual inhibitor of Bcl-2 and SERCA proteins, sHA 14-1, with selective cytotoxicity toward MDR cancer cell lines in vitro. In this study, we present new evidence for its therapeutic potential in treatment of MDR cancers and offer mechanistic insights toward its preferential targeting of drug-resistant cancer. CXL017 selectively suppressed the growth of tumors derived from the MDR cancer cell line, HL60/MX2, in vivo. In addition, even after chronic exposure to CXL017, HL60/MX2 failed to develop stable resistance to CXL017, whereas it acquired >2000-fold resistance to cytarabine (Ara-C), the major first-line chemotherapy for the treatment of acute myeloid leukemia (AML). Remarkably, instead of acquiring further cross-resistance, HL60/MX2 cells exposed to CXL017 were resensitized to standard therapies (10- to 100-fold). Western blotting analyses revealed that CXL017 exposure significantly down-regulated Mcl-1 and Bax and up-regulated Noxa, Bim, Bcl-X(L), SERCA2, and SERCA3 proteins, along with a reduction in endoplasmic reticulum (ER) calcium content. Given the well-established functions of Bcl-2 family proteins and ER calcium in drug resistance, our results suggest that the down-regulation of Mcl-1 and the up-regulation of Noxa and Bim along with the decrease in ER calcium content are likely responsible for CXL017-induced resensitization of MDR cancer cells. These data also demonstrate the unique potential of CXL017 to overcome MDR in cancer treatment.

Abstract 767: CXL017 re-sensitizes multidrug resistant leukemia cells to chemotherapy via modulating Bcl-2 family proteins and SERCA proteins

Abstract Multidrug resistance (MDR) is a major hurdle in the treatment of cancer and there is a pressing need for new therapies. We have recently developed CXL017, derived from a Bcl-2 and SERCA dual inhibitor, with selective cytotoxicity towards MDR cancer cells. In this study, we present new evidence for its therapeutic potential in treatment of MDR cancers and offer mechanistic insights. CXL017 significantly and selectively suppresses the growth of tumors derived from the MDR cancer cell line, HL60/MX2, in vivo. In addition, HL60/MX2 failed to develop stable resistance to CXL017 upon prolonged exposure. In comparison, HL60/MX2 acquired greater than 2000-fold resistance to cytarabine (Ara-C). Remarkably, instead of acquiring cross-resistance, HL60/MX2 cells exposed to CXL017 were re-sensitized to standard therapies (10 to 100-fold). Western blotting analyses revealed that HL60/MX2 has significantly elevated levels of Mcl-1, Bax and Bak relative to HL60. HL60/MX2 also over-expresses SERCA2 and SERCA3 with elevated endoplasmic reticulum (ER) calcium content. CXL017 exposure significantly down-regulates Mcl-1 and Bax but causes up-regulation of Bcl-XL, SERCA2, and SERCA3 proteins, along with a reduction in ER calcium content. Given the well-established functions of Bcl-2 family proteins and ER calcium in drug resistance, our results suggest that the reduction in Mcl-1 and the decrease in ER calcium content are responsible for CXL017 induced chemosensitization of MDR cancer cells. These results support the strategy to develop dual inhibitors of anti-apoptotic Bcl-2 proteins and SERCA to treat MDR cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 767. doi:1538-7445.AM2012-767

Crystal Structure of a BCL-W Domain-Swapped Dimer: Implications for the Function of BCL-2 Family Proteins

The prosurvival and proapoptotic proteins of the BCL-2 family share a similar three-dimensional fold despite their opposing functions. However, many biochemical studies highlight the requirement for conformational changes for the functioning of both types of proteins, although structural data to support such changes remain elusive. Here, we describe the X-ray structure of dimeric BCL-W that reveals a major conformational change involving helices α3 and α4 hinging away from the core of the protein. Biochemical and functional studies reveal that the α4-α5 hinge region is required for dimerization of BCL-W, and functioning of both pro- and antiapoptotic BCL-2 proteins. Hence, this structure reveals a conformational flexibility not seen in previous BCL-2 protein structures and provides insights into how these regulators of apoptosis can change conformation to exert their function.

Targeting the BCL-2 family in malignancies of germinal centre origin

The germinal centre is a dynamic microenvironment where B-cell responses are honed. Antigen-specific cells undergo clonal expansion followed by antibody affinity maturation and class switching through somatic hypermutation and recombination of immunoglobulin genes respectively. The huge proliferative capacity of the B-cells and the potential for generating non-functional or autoreactive immunoglobulins, necessitate strict control measures. Pro-apoptotic signalling pathways via B-cell receptors, FAS and the TGF-β receptor, ALK5, ensure that apoptosis of germinal centre B-cells is the norm and cells only survive to differentiate fully if they receive sufficient pro-survival signals to overcome their ‘primed for death’ status. Several of the B-cell signalling pathways converge on the intrinsic apoptotic machinery to control expression of the BCL-2 family of apoptosis regulators including BIM, the pro-survival factor BCL-XL and the BH3-only protein, BIK (recently identified as a mediator of a TGF-β-induced default apoptosis pathway in human B-cells). It is a foreseeable hazard that cells undergoing genetic mutation and recombination events might unintentionally target some of these factors, resulting in defective programmed cell death. Here we discuss the function of BCL-2 family proteins in germinal centre reactions, their deregulation in malignancies of germinal centre origin, and the potential for targeting BCL-2-related proteins therapeutically in lymphomas.

Dual Mechanisms of sHA 14-1 in Inducing Cell Death through Endoplasmic Reticulum and Mitochondria

HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Delta psi m) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Delta psi m and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.

Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.

ApoG2, a novel inhibitor of antiapoptotic Bcl‐2 family proteins, induces apoptosis and suppresses tumor growth in nasopharyngeal carcinoma xenografts

Nasopharyngeal carcinoma (NPC) is a common malignant tumor in South China. It has been reported that overexpression of antiapoptotic Bcl-2 family proteins in NPC has caused the lack of long-term efficacy of conventional therapies. Apogossypolone (ApoG2), a novel small-molecule inhibitor of antiapoptotic Bcl-2 family proteins, has been discovered as the optimized derivative of gossypol. In this study, we found that in NPC cells, ApoG2 totally blocked the antiapoptotic function of Bcl-2 family proteins without affecting the expression levels of these proteins. ApoG2 selectively inhibited proliferation of 3 NPC cell lines (C666-1, CNE-1 and CNE-2) that highly expressed the antiapoptotic Bcl-2 proteins. This inhibitory activity was associated with release of cytochrome c, activation of caspase-9 and caspase-3 and apoptosis of sensitive NPC cells. However, ApoG2 had no obvious inhibitory effect on NPC cell line HONE-1, which expressed antiapoptotic Bcl-2 and Bcl-xL at a low level. We further found that ApoG2 effectively suppressed tumor growth of NPC xenografts in nude mice and enhanced the antitumor effect of CDDP (cisplatin) on NPC cells in vitro and in vivo. Immunohistochemical results showed that the expression of CD31 decreased after ApoG2 treatment, which suggested inhibition of angiogenesis in NPC xenografts. Our findings strongly suggest that ApoG2 may serve as a novel inhibitor of Bcl-2 family proteins and, by targeting these proteins, may become a promising drug for the treatment of NPC.