Bcl-2 Homology Research Articles

The pro-apoptotic function of the C. elegans BCL-2 homolog CED-9 requires interaction with the APAF-1 homolog CED-4.

In Caenorhabditis elegans, apoptosis is inhibited by the BCL-2 homolog CED-9. Although canonically anti-apoptotic, CED-9 has a poorly understood pro-apoptotic function. CED-9 is thought to inhibit apoptosis by binding to and inhibiting the pro-apoptotic C. elegans APAF-1 homolog CED-4. We show that CED-9 or CED-4 mutations located in their CED-9-CED-4 binding regions reduce apoptosis without affecting the CED-9 anti-apoptotic function. These mutant CED-9 and CED-4 proteins are defective in a CED-9-CED-4 interaction in vitro and in vivo, revealing that the known CED-9-CED-4 interaction is required for the pro-apoptotic but not for the anti-apoptotic function of CED-9. The pro-apoptotic CED-9-CED-4 interaction occurs at mitochondria. In mammals, BCL-2 family members can activate APAF-1 via cytochrome c release from mitochondria. The conserved role of mitochondria in CED-9/BCL-2-dependent CED-4/APAF-1 activation is notable and suggests that understanding how CED-9 promotes apoptosis in C. elegans could inform the understanding of mammalian apoptosis and how disruptions of apoptosis promote certain human disorders.

Bcl-2 Orthologues, Buffy and Debcl, Can Suppress Drp1-Dependent Age-Related Phenotypes in Drosophila.

The relationship of Amyotrophic Lateral Sclerosis, Parkinson's disease, and other age-related neurodegenerative diseases with mitochondrial dysfunction has led to our study of the mitochondrial fission gene Drp1 in Drosophila melanogaster and aspects of aging. Previously, the Drp1 protein has been demonstrated to interact with the Drosophila Bcl-2 mitochondrial proteins, and Drp1 mutations can lead to mitochondrial dysfunction and neuronal loss. In this study, the Dopa decarboxylase-Gal4 (Ddc-Gal4) transgene was exploited to direct the expression of Drp1 and Drp1-RNAi transgenes in select neurons. Here, the knockdown of Drp1 seems to compromise locomotor function throughout life but does not alter longevity. The co-expression of Buffy suppresses the poor climbing induced by the knockdown of the Drp1 function. The consequences of Drp1 overexpression, which specifically reduced median lifespan and diminished climbing abilities over time, can be suppressed through the directed co-overexpression of pro-survival Bcl-2 gene Buffy or by the co-knockdown of the pro-cell death Bcl-2 homologue Debcl. Alteration of the expression of Drp1 acts to phenocopy neurodegenerative disease phenotypes in Drosophila, while overexpression of Buffy can counteract or rescue these phenotypes to improve overall health. The diminished healthy aging due to either the overexpression of Drp1 or the RNA interference of Drp1 has produced novel Drosophila models for investigating mechanisms underlying neurodegenerative disease.

Dissecting the neuroprotective interaction between the BH4 domain of BCL-w and the IP3 receptor

BCL-w is a BCL-2 family protein that promotes cell survival in tissue- and disease-specific contexts. The canonical anti-apoptotic functionality of BCL-w is mediated by a surface groove that traps the BCL-2 homology 3 (BH3) α-helices of pro-apoptotic members, blocking cell death. A distinct N-terminal portion of BCL-w, termed the BCL-2 homology 4 (BH4) domain, selectively protects axons from paclitaxel-induced degeneration by modulating IP3 receptors, a noncanonical BCL-2 family target. Given the potential of BCL-w BH4 mimetics to prevent or mitigate chemotherapy-induced peripheral neuropathy, we sought to characterize the interaction between BCL-w BH4 and the IP3 receptor, combining "staple" and alanine scanning approaches with molecular dynamics simulations. We generated and identified stapled BCL-w BH4 peptides with optimized IP3 receptor binding and neuroprotective activities. Point mutagenesis further revealed the sequence determinants for BCL-w BH4 specificity, providing a blueprint for therapeutic targeting of IP3 receptors to achieve neuroprotection.

Profiling protein-protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia.

B-cell-lymphoma-2 (BCL2) homology-3 (BH3) mimetics are inhibitors of protein-protein interactions (PPIs) that saturate anti-apoptotic proteins in the BCL2 family to induce apoptosis in cancer cells. Despite the success of the BH3-mimetic ABT-199 for the treatment of haematological malignancies, only a fraction of patients respond to the drug and most patients eventually develop resistance to it. Here we show that the efficacy of ABT-199 can be predicted by profiling the rewired status of the PPI network of the BCL2 family via single-molecule pull-down and co-immunoprecipitation to quantify more than 20 types of PPI from a total of only 1.2 × 106 cells per sample. By comparing the obtained multidimensional data with BH3-mimetic efficacies determined ex vivo, we constructed a model for predicting the efficacy of ABT-199 that designates two complexes of the BCL2 protein family as the primary mediators of drug effectiveness and resistance, and applied it to prospectively assist therapeutic decision-making for patients with acute myeloid leukaemia. The characterization of PPI complexes in clinical specimens opens up opportunities for individualized protein-complex-targeting therapies.

Modeling the Binding of Anticancer Peptides and Mcl-1.

Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of pro-apoptotic proteins have been demonstrated to bind the hydrophobic groove of anti-apoptotic Mcl-1, and this interaction is responsible for regulating apoptosis. Structural studies have shown that, while there is high overall structural conservation among the anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins, differences in the surface groove of these proteins facilitates binding specificity. This binding specificity is crucial for the mechanism of action of the Bcl-2 family in regulating apoptosis. Bim-based peptides bind specifically to the hydrophobic groove of Mcl-1, emphasizing the importance of these interactions in the regulation of cell death. Molecular docking was performed with BH3-like peptides derived from Bim to identify high affinity peptides that bind to Mcl-1 and to understand the molecular mechanism of their interactions. The interactions of three identified peptides, E2gY, E2gI, and XXA1_F3dI, were further evaluated using 250 ns molecular dynamics simulations. Conserved hydrophobic residues of the peptides play an important role in their binding and the structural stability of the complexes. Understanding the molecular basis of interaction of these peptides will assist in the development of more effective Mcl-1 specific inhibitors.

Dysregulation of BCL-2 family proteins in blood neoplasm: therapeutic relevance of antineoplastic agent venetoclax

During cellular stress, the master regulators of intrinsic self-death (apoptosis) are BCL-2 family proteins. The BCL-2 family proteins play a key role in apoptosis and are tightly regulated via other BCL-2 family proteins, non-BCL-2 protein suppressors, and epigenetic modifications. As the name implies, these proteins possess one or two of the four BCL-2 homology domains (BH1–BH4). According to their roles, they are classified as pro-apoptotic or pro-survival proteins. BH-3-only proteins possess a single BH3 domain and are specific/key effector proteins for intracellular death commitment, particularly in the context of cell survival and programmed cell death. This delicate interplay among the BCL-2 family members is essential for maintaining the primary hemostasis, or balance, of cell fate. The anti-apoptotic proteins, such as BCL-2 and BCL-XL, promote cell survival by inhibiting apoptosis. On the other hand, the pro-apoptotic proteins, such as BAX and BAK, drive apoptosis. It ensures that cells are able to respond appropriately to various internal and external signals, ultimately determining whether a cell survives or undergoes programmed cell death. Understanding and targeting this delicate balance is a promising avenue for developing therapeutic strategies to modulate cell fate and treat various diseases. The molecular pathogenesis of BCL-2 family proteins in blood disorders involves differential expression of these components resulting in the dysregulation of the pathway contributing to cell survival and resistance to apoptosis as observed in follicular lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, and acute myeloid leukemia. Such dysregulation is a major impediment to standard therapies and aids in chemo resistance. Studies show some promising clinical outcomes with antineoplastic agent venetoclax either as a monotherapy or in combination with other agents. This review discusses recent studies on the regulation of BCL-2 family proteins which might provide a molecular landscape for their clinical implications in blood disorders.

Identification and function analysis of BCL2 in immune response of Pteria penguin

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.

Venetoclax-Related Neutropenia in Leukemic Patients: A Comprehensive Review of the Underlying Causes, Risk Factors, and Management.

Venetoclax is a Bcl-2 homology domain 3 (BH3) mimetic currently approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) that has proven to be highly effective in reinstating apoptosis in leukemic cells through the highly selective inhibition of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). Clinically, venetoclax has provided lasting remissions through the inhibition of CLL and AML blasts. However, this activity has often come at the cost of grade III/IV neutropenia due to hematopoietic cells' dependence on Bcl-2 for survival. As life-threatening infections are an important complication in these patients, an effective management of neutropenia is indispensable to maximize patient outcomes. While there is general consensus over dose reduction and scheduling modifications to minimize the risk of neutropenia, the impact of these modifications on survival is uncertain. Moreover, guidelines do not yet adequately account for patient-specific and disease-specific risk factors that may predict toxicity, or the role combination treatment plays in exacerbating neutropenia. The objective of this review is to discuss the venetoclax-induced mechanism of hematological toxicity, the potential predictive risk factors that affect patient vulnerability to neutropenia, and the current consensus on practices for management of neutropenia.

Targeted intracellular delivery of BH3 mimetic peptide inhibits BCL-2 activity and prevents breast cancer development

<p class="MsoNormal" style="margin-top: 12pt; text-align: justify;"><span lang="EN-US" style="font-family: Cambria, serif; font-size: 14pt;">Breast cancer, as a malignant tumor with easy metastasis and poor prognosis, threatens the health of women around the world. Increasing studies have shown that the Bcl-2 family of apoptosis-related proteins is often expressed abnormally in breast cancer. The Bcl-2 homology 3 (BH3) mimetic peptide can bind and neutralize Bcl-2, preventing its binding to the apoptosis "effector" proteins Bak and Bax, thereby promoting the apoptosis process. However, there is a lack of effective intracellular delivery system for BH3 to exert its biological activity. Therefore, this study utilized an activatable supercharged polypeptide (ASCP) tumor-targeted delivery platform based on pH and protease response to achieve the targeted release of BH3 at the tumor site. Ultimately, intracellular delivery of BH3 was achieved and induced apoptosis of breast tumor cells, preventing the development of breast cancer.</span></p>

Effective Targeting of Melanoma Cells by Combination of Mcl-1 and Bcl-2/Bcl-xL/Bcl-w Inhibitors.

Recent advances in melanoma therapy have significantly improved the prognosis of metastasized melanoma. However, large therapeutic gaps remain that need to be closed by new strategies. Antiapoptotic Bcl-2 proteins critically contribute to apoptosis deficiency and therapy resistance. They can be targeted by BH3 mimetics, small molecule antagonists that mimic the Bcl-2 homology domain 3 (BH3) of proapoptotic BH3-only proteins. By applying in vitro experiments, we aimed to obtain an overview of the possible suitability of BH3 mimetics for future melanoma therapy. Thus, we investigated the effects of ABT-737 and ABT-263, which target Bcl-2, Bcl-xL and Bcl-w as well as the Bcl-2-selective ABT-199 and the Mcl-1-selective S63845, in a panel of four BRAF-mutated and BRAF-WT melanoma cell lines. None of the inhibitors showed significant effectiveness when used alone; however, combination of S63845 with each one of the three ABTs almost completely abolished melanoma cell survival and induced apoptosis in up to 50-90% of the cells. Special emphasis was placed here on the understanding of the downstream pathways involved, which may allow improved applications of these strategies. Thus, cell death induction was correlated with caspase activation, loss of mitochondrial membrane potential, phosphorylation of histone H2AX, and ROS production. Caspase dependency was demonstrated by a caspase inhibitor, which blocked all effects. Upregulation of Mcl-1, induced by S63845 itself, as reported previously, was blocked by the combinations. Indeed, Mcl-1, as well as XIAP (X-linked inhibitor of apoptosis), were strongly downregulated by combination treatments. These findings demonstrate that melanoma cells can be efficiently targeted by BH3 mimetics, but the right combinations have to be selected. The observed pronounced activation of apoptosis pathways demonstrates the decisive role of apoptosis in the loss of cell viability by BH3 mimetics.

Development of recombinant protein-based nanoparticle systems for inducing tumor cell apoptosis: In vitro evaluation of their cytotoxic and apoptotic effects on cancer cells

Myeloid leukemia cell differentiation protein (Mcl-1) is a B-cell lymphoma 2 (Bcl-2) family antiapoptotic protein that regulates cellular apoptosis. Mcl-1 expression is important for cancer cell survival and a prospective target in cancer treatment. Bcl-2 homology 3 (BH3) mimetic peptides, such as the Mcl-1-specific MS1 peptide that targets conserved BH3 domains, are highly specific Mcl-1 inhibitors. However, the application of these peptides is limited due to their metabolic instability, degradation by cellular proteases, and low cell permeability. In this study, it was aimed to form nanostructures to increase the cellular uptake and stability of the Mcl-1 inhibitor MS1 peptide. The Bim BH3 mimetic MS1 peptide was produced recombinantly in fusion with mNeonGreen fluorescent protein and polyhistidine (6xHisTag). Self-assembled protein nanoparticles were fabricated from the recombinant MS1-mNeonGreen protein. Furthermore, the MS1-mNeonGreen protein was encapsulated into polymeric nanoparticles. The cytotoxic effects of these nanoparticles on cancer cells [cervical cancer cell line (HeLa), lung cancer cell line (A549) and breast cancer cell line (MDA-MB-231)] and healthy cell line [Human umbilical vein endothelial cells (HUVEC)] was analyzed by MTT test. The apoptotic effect of nanoparticles on these cells was investigated by determining the antiapoptotic and proapoptotic protein levels. As a result, it was revealed that the nanoparticles have a specific cytotoxic effect against the cells and induce apoptosis.

Phase 1 trial of navitoclax and sorafenib in patients with refractory solid tumors with a hepatocellular carcinoma expansion cohort.

507 Background: Navitoclax (ABT-263) is an oral Bcl-2 homology-3 mimetic that binds with high affinity to pro-survival Bcl-2 proteins, resulting in apoptosis. Sorafenib, an oral multi kinase inhibitor inhibits tumor angiogenesis and also promotes apoptosis. The efficacy of either agent alone is limited; however, preclinical studies demonstrate synergy with the combination of navitoclax and sorafenib. Methods: In this phase 1 pharmacokinetic and pharmacodynamic study (NCT01364051), we evaluated the combination of navitoclax and sorafenib in a dose escalation cohort of patients with refractory solid tumors, with an expansion cohort in hepatocellular carcinoma (HCC). The study agents were provided by the Cancer Therapy Evaluation Program at National Cancer Institute under CRADA. Navitoclax and sorafenib were administered continuously on days 1 through 21 of 21-day cycles. The maximum tolerated dose (MTD) was determined using the continual reassessment method. Results: Ten patients were enrolled in the dose escalation cohort and 15 patients with HCC were enrolled in the expansion cohort. Two dose levels were tested, dose level 1 (navitoclax 150 mg daily plus sorafenib 400 mg twice daily) and dose level 2 (navitoclax 200 mg daily plus sorafenib 400 mg twice daily). At dose level 1, one dose limiting toxicity (DLT) [grade 3 aspartate aminotransferase elevation] was observed among 6 patients treated at this dose level. In a cohort of 3 patients treated at dose level 2, two DLTs were observed (grade 3 hypertension, and grade 3 rash), hence dose level 1 was identified as the MTD. Among all patients, the most common grade 3 toxicity was thrombocytopenia (5 patients, 20%): there were no grade 4 or 5 toxicities (Table). Patients received a median of 2 cycles (range 1-36 cycles) and all patients were off study treatment at data cut off. Six patients in the expansion cohort had stable disease, 5 had progressive disease and 4 with unavailable data. There were no partial or complete responses. Drug-drug interaction between navitoclax and sorafenib was not observed. The combination of navitoclax and sorafenib did not increase induction of apoptosis compared with navitoclax alone. Conclusions: Navitoclax plus sorafenib resulted in manageable, but not ideal toxicities for continuous oral administration which precluded dose escalation. The lack of clinical benefit in the HCC expansion cohort does not support further development of this combination for the treatment of advanced HCC. Clinical trial information: NCT01364051 . [Table: see text]

Non-canonical BIM-regulated energy metabolism determines drug-induced liver necrosis

Paracetamol (acetaminophen, APAP) overdose severely damages mitochondria and triggers several apoptotic processes in hepatocytes, but the final outcome is fulminant necrotic cell death, resulting in acute liver failure and mortality. Here, we studied this switch of cell death modes and demonstrate a non-canonical role of the apoptosis-regulating BCL-2 homolog BIM/Bcl2l11 in promoting necrosis by regulating cellular bioenergetics. BIM deficiency enhanced total ATP production and shifted the bioenergetic profile towards glycolysis, resulting in persistent protection from APAP-induced liver injury. Modulation of glucose levels and deletion of Mitofusins confirmed that severe APAP toxicity occurs only in cells dependent on oxidative phosphorylation. Glycolytic hepatocytes maintained elevated ATP levels and reduced ROS, which enabled lysosomal recycling of damaged mitochondria by mitophagy. The present study highlights how metabolism and bioenergetics affect drug-induced liver toxicity, and identifies BIM as important regulator of glycolysis, mitochondrial respiration, and oxidative stress signaling.

B-cell lymphoma-2 family proteins in the crosshairs: Small molecule inhibitors and activators for cancer therapy.

The B-cell lymphoma-2 (BCL-2) family of proteins plays a crucial role in the regulation of apoptosis, offering a dual mechanism for its control. Numerous studies have established a strong association between gene disorders of these proteins and the proliferation of diverse cancer cell types. Consequently, the identification and development of drugs targeting BCL-2 family proteins have emerged as a prominent area in antitumor therapy. Over the last two decades, several small-molecules have been designed to modulate the protein-protein interactions between anti- and proapoptotic BCL-2 proteins, effectively suppressing tumor growth and metastasis in vivo. The primary focus of research has been on developing BCL-2 homology 3 (BH3) mimetics to target antiapoptotic BCL-2 proteins, thereby competitively releasing proapoptotic BCL-2 proteins and restoring the blocked intrinsic apoptotic program. Additionally, for proapoptotic BCL-2 proteins, exogenous small molecules have been explored to activate cell apoptosis by directly interacting with executioner proteins such as BCL-2-associated X protein (BAX) or BCL-2 homologous antagonist/killer protein (BAK). In this comprehensive review, we summarize the inhibitors and activators (sensitizers) of BCL-2 family proteins developed over the past decades, highlighting their discovery, optimization, preclinical and clinical status, and providing an overall landscape of drug development targeting these proteins for therapeutic purposes.

Invariant BECN1 CXXC motifs bind Zn2+ and regulate structure and function of the BECN1 intrinsically disordered region

ABSTRACT Autophagy is an essential catabolic cellular homeostasis process conserved in all eukaryotes. BECN1/Beclin 1, a key autophagy protein involved in autophagosome nucleation, contains two invariant CxxC motifs within a large intrinsically disordered region (IDR) at the BECN1 N terminus. We used inductively coupled plasma mass spectrometry to demonstrate that BECN1 binds Zn2+ in a 1:1 molar ratio, and that mutation of the invariant cysteines abrogated Zn2+ binding, demonstrating that the invariant CxxC motifs were responsible for binding Zn2+. We used circular dichroism spectroscopy to show that Zn2+-binding increases IDR helicity, and likely also binding-associated helical transitions. Further, we used small angle X-ray scattering and multi-angle light scattering to show that Zn2+-binding made the BECN1 IDR more compact, and HDX-MS to show that the Zn2+-bound state of the IDR had reduced solvent accessibility. We also used isothermal titration calorimetry to show that the BECN1 IDR did not self-associate and abrogation of Zn2+ binding does not affect binding of a viral BCL2 homolog. Last, we showed that both CxxC motifs are required, but the intervening IDR is less important, for starvation-triggered upregulation of autophagy. Thus, Zn2+-binding via the CxxC motifs likely facilitates binding-associated helical conformational transitions of BECN1 important for up-regulating BECN1-mediated autophagy. Abbreviations AFM: aromatic finger mutant; BH3D: BCL2 homology 3 domain; CCD: coiled-coil domain; CD: circular dichroism spectroscopy; [CysDM1]: C18S and C21S double mutant; [CysDM2]: C137S, and C140S double mutant; [CysTM], C18S, C21S, C137S, and C140S tetrad mutant; Dmax: maximum particle diameter; dRI, differential refractive index; EFA: evolving factor analysis; FHD: flexible helical domain; FL: full length; GFP: green fluorescent protein; HDX-MS: hydrogen/deuterium exchange mass spectrometry; ICP-MS: inductively coupled plasma mass spectrometry; IDR: intrinsically disordered region; ITC, isothermal titration calorimetry; MALS, multi angle light scattering; MBP: maltose-binding protein; MoRFs: molecular recognition features; P(r): pairwise-distance distribution; PtdIns3K: class III phosphatidylinositol 3-kinase; Rg: radius of gyration; SASBDB: small angle scattering biological data bank; SEC: size-exclusion chromatography; SEC-SAXS: size-exclusion chromatography in tandem with small angle X-ray scattering; TEV: tobacco-etch virus; TFE: 2,2,2-trifluoroethanol; TPEN: N,N,N,N-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine; Vc: volume of correlation; WT: wild-type.

Epstein-Barr Virus Encoded BCL2, BHRF1, Downregulates Autophagy by Noncanonical Binding of BECN1.

γ-herpesviruses (γHVs) encode BCL2 homologues (vBCL2) that bind the Bcl-2 homology 3 domains (BH3Ds) of diverse proteins, inhibiting apoptosis and promoting host cell and virus survival. vBCLs encoded by Kaposi sarcoma-associated HV (KSHV) and γHV68 downregulate autophagy, a degradative cellular process crucial for homeostasis and innate immune responses to pathogens, by binding to a BH3D in BECN1, a key autophagy protein. Epstein-Barr virus (EBV) encodes a vBCL2 called BHRF1. Here we show that unlike the KSHV and γHV68 vBCL2s, BHRF1 does not bind the isolated BECN1 BH3D. We use yeast two-hybrid assays to identify the minimal region of BECN1 required and sufficient for binding BHRF1. We confirm that this is a direct, albeit weak, interaction via affinity pull-down assays and isothermal titration calorimetry. To understand the structural bases of BHRF1 specificity, we determined the 2.6 Å crystal structure of BHRF1 bound to the BID BH3D, which binds ∼400-times tighter to BHRF1 than does BECN1, and performed a detailed structural comparison with complexes of diverse BH3Ds bound to BHRF1 and to other antiapoptotic BCL2s. Lastly, we used mammalian cell autophagy assays to demonstrate that BHRF1 downregulates autophagy and that a cell-permeable peptide derived from the BID BH3D inhibits BHRF1-mediated downregulation of autophagy. In summary, our results suggest that BHRF1 downregulates autophagy by noncanonical binding of a flexible region of BECN1 that includes but is not limited to the BH3D and that BH3D-derived peptides that bind better to BHRF1 can block downregulation of autophagy by BHRF1.

Integrated epigenetic and transcriptional single-cell analysis of t(11;14) multiple myeloma and its BCL2 dependency

AbstractThe translocation t(11;14) occurs in 20% of patients with multiple myeloma (MM) and results in the upregulation of CCND1. Nearly two-thirds of t(11;14) MM cells are BCL2 primed and highly responsive to the oral BCL2 inhibitor venetoclax. Although it is evident that this unique sensitivity to venetoclax depends on the Bcl-2 homology domain 3– proapoptotic protein priming of BCL2, the biology underlying t(11;14) MM dependency on BCL2 is poorly defined. Importantly, the epigenetic regulation of t(11;14) transcriptomes and its impact on gene regulation and clinical response to venetoclax remain elusive. In this study, by integrating assay for transposase-accessible chromatin by sequencing (ATAC-seq) and RNA-seq at the single-cell level in primary MM samples, we have defined the epigenetic regulome and transcriptome associated with t(11;14) MM. A B-cell–like epigenetic signature was enriched in t(11;14) MM, confirming its phylogeny link to B-cell rather than plasma cell biology. Of note, a loss of a B-cell–like epigenetic signature with a gain of canonical plasma cell transcription factors was observed at the time of resistance to venetoclax. In addition, MCL1 and BCL2L1 copy number gains and structural rearrangements were linked to venetoclax resistance in patients with t(11;14) MM. To date, this is the first study in which both single-cell (sc) ATAC-seq and scRNA-seq analysis are integrated into primary MM cells to obtain a deeper resolution of the epigenetic regulome and transcriptome associated with t(11;14) MM biology and venetoclax resistance.

The BCL-2 family protein BCL-RAMBO interacts and cooperates with GRP75 to promote its apoptosis signaling pathway

The BCL-2 family protein BCL-RAMBO, also known as BCL2-like 13, anchors at the outer mitochondrial membrane and regulates apoptosis, mitochondrial fragmentation, and mitophagy. However, the mechanisms underlying the proapoptotic role of BCL-RAMBO remain unclear. In the present study, we demonstrated that BCL-RAMBO interacted with glucose-regulated protein 75 (GRP75), also known as heat shock protein family A member 9, and mortalin using co-immunoprecipitation and glutathione S-transferase-based pull-down assays. BCL-RAMBO interacted with GRP75 via its No BCL-2 homology domain. The interaction between BCL-RAMBO and GRP75 was confirmed by genetic interactions in Drosophila because a rough eye phenotype caused by the ectopic expression of BCL-RAMBO was partially suppressed by mutations in Hsc70-5, a mammalian GRP75 ortholog. In human embryonic kidney 293T cells, the co-expression of BCL-RAMBO and GRP75 facilitated an elevation in executioner caspase activity and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage. In contrast, the knockdown of GRP75 suppressed elevated executioner caspase activity and PARP-1 cleavage in BCL-RAMBO-transfected cells. The mitochondrial release of cytochrome c induced by BCL-RAMBO was also attenuated by the knockdown of GRP75. These results indicate that GRP75 interacts with BCL-RAMBO and plays a crucial role in the BCL-RAMBO-dependent apoptosis signaling pathway.

Crystal structure of Bak bound to the BH3 domain of Bnip5, a noncanonical BH3 domain-containing protein.

The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.

Targeting MCL-1 protein to treat cancer: opportunities and challenges.

Evading apoptosis has been linked to tumor development and chemoresistance. One mechanism for this evasion is the overexpression of prosurvival B-cell lymphoma-2 (BCL-2) family proteins, which gives cancer cells a survival advantage. Mcl-1, a member of the BCL-2 family, is among the most frequently amplified genes in cancer. Targeting myeloid cell leukemia-1 (MCL-1) protein is a successful strategy to induce apoptosis and overcome tumor resistance to chemotherapy and targeted therapy. Various strategies to inhibit the antiapoptotic activity of MCL-1 protein, including transcription, translation, and the degradation of MCL-1 protein, have been tested. Neutralizing MCL-1's function by targeting its interactions with other proteins via BCL-2 interacting mediator (BIM)S2A has been shown to be an equally effective approach. Encouraged by the design of venetoclax and its efficacy in chronic lymphocytic leukemia, scientists have developed other BCL-2 homology (BH3) mimetics-particularly MCL-1 inhibitors (MCL-1i)-that are currently in clinical trials for various cancers. While extensive reviews of MCL-1i are available, critical analyses focusing on the challenges of MCL-1i and their optimization are lacking. In this review, we discuss the current knowledge regarding clinically relevant MCL-1i and focus on predictive biomarkers of response, mechanisms of resistance, major issues associated with use of MCL-1i, and the future use of and maximization of the benefits from these agents.