Bcl-2 Homology Research Articles

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.

O-159 Impact of open and closed methods of vitrification on oocyte mitochondria function and RNAseq transcriptome profile: a preclinical screening study

Abstract Study question Do current methods of open vitrification (OV) and closed vitrification (CV) impact on oocyte mitochondria function and transcriptome? Summary answer Oocyte vitrification confers unique mitochondria distribution patterns and alters the expression of stress response genes and mitochondria function relative to control (fresh) oocytes. What is known already Mitochondria are essential for maintenance of metabolic function and developmental competence at physiological conditions. Conversely, open and closed methods of oocyte vitrification are associated with physical, chemical and non-physiological changes, and exposures to cryoprotecting agents which could be harmful at extremely high and low temperatures. Study design, size, duration Ninety-four unfertilised Metaphase II (MII) oocytes were donated to research between May 2018 and August 2021. Fifty-seven oocytes were randomly allocated to the different study groups. Similarly, thirty-seven oocytes were allocated to the different study control groups. Participants/materials, setting, methods Fourteen women underdoing IVF/ICSI treatment cycles were involved in the study. cDNA was obtained for qPCR and the Smart-Seq2 protocol and single-cell RNA-Seq was performed (illumina NextSeq2000). Mitochondrial staining on live-oocytes was performed using Mito-tracker Deep Red stain, and images evaluated using fluorescence microscopy. Differentially expressed genes were identified using the R package edgeR and functional gene ontology was performed using an over-representation analysis via WebGestalt. Correction for multiple testing was calculated using Benjamini-Hochberg method Main results and the role of chance Mean mitochondrial intensity/density showed similarities between open (OV) and closed vitrification (CV) and control (fresh oocytes FO): 25.8 (12.7-44.9, standard deviation [SD] 10.2) for FO oocytes (n = 15), 30.3 (16.2-44.8, SD 10.1) for OV oocytes (n = 11), 26.6 (13.3-64.6, SD 15.3) for CV oocytes (n = 12) (p = 0.617). Distinct mitochondria distribution patterns (cytoplasmic and peripheral) were associated with OV and CV oocytes. Twice as many peripheral to cytoplasmic patterns were observed in FO and OV oocytes in contrast to CV oocytes (p = 037). Mean expression of the gene SDHB, which is associated with the TCA cycle and the mitochondrial electron transport chain was reduced in vitrified oocytes (n = 13) (0.0879 ± 0.0216 vs 0.0593 ± 0.0479), as was the anti-apoptotic gene BCL2 (p = 0.0061), relative to control oocytes (n = 13), (p = 0.01). Conversely, genes associated with apoptosis and stress response (BAX (p = 0.002) and BCL2L1 (p = 0.016)) were significantly enriched relative to control oocytes. RNAseq transcriptome analysis revealed a total of 71 differentially expressed genes following vitrification relative to control oocytes. Genes associated with critical metabolic processes (death domain binding, BCL-2 homology 3 (BH3) domains, and exopolyphosphatatse activities) were enriched in vitrified oocytes relative to the control group (FDR>0.05). Limitations, reasons for caution The use of unfertilised metaphase II (MII) oocytes on day 1 as a preclinical application screening model for vitrification may not reflect the molecular status and integrity of clinical grade MII oocytes on day 0. Wider implications of the findings This study emphasizes the inherent impact of vitrification on oocyte mitochondria function and transcriptome relative to control oocytes. Importantly, unintended perturbations may be detrimental to oocyte health and developmental competence, suggesting the need for a continuous safety screening and monitoring of ART methods of cryopreservation and offspring long-term health. Trial registration number National Institute for Health Research (NIHR). Grant Reference: ICA-CDRF-2015-01-068

Mitophagy associated self-degradation of phosphorylated MAP4 guarantees the migration and proliferation responses of keratinocytes to hypoxia

Our previous study has announced that phosphorylated microtubule-associated protein 4 (p-MAP4) accelerated keratinocytes migration and proliferation under hypoxia through depolymerizing microtubules. However, p-MAP4 should exhibit inhibitory effects on wound healing, for it also impaired mitochondria. Thus, figuring out the outcome of p-MAP4 after it impaired mitochondria and how the outcome influenced wound healing were far-reaching significance. Herein, the results revealed that p-MAP4 might undergo self-degradation through autophagy in hypoxic keratinocytes. Next, p-MAP4 activated mitophagy which was unobstructed and was also the principal pathway of its self-degradation triggered by hypoxia. Moreover, both Bcl-2 homology 3 (BH3) and LC3 interacting region (LIR) domains had been verified in MAP4, and they endowed MAP4 with the capability to synchronously function as a mitophagy initiator and a mitophagy substrate receptor. And, mutating any one of them ruined hypoxia-induced self-degradation of p-MAP4, resulting in destroyed proliferation and migration responses of keratinocytes to hypoxia. Our findings unviewed that p-MAP4 experienced mitophagy-associated self-degradation through utilizing its BH3 and LIR domains under hypoxia. As a result, the mitophagy-associated self-degradation of p-MAP4 guaranteed the migration and proliferation responses of keratinocytes to hypoxia. Together, this research provided a bran-new pattern of proteins in regulating wound healing, and offered a new direction for intervening wound healing.

Apoptotic gene loss in Cnidaria is associated with transition to parasitism

The phylum Cnidaria consists of several morphologically diverse classes including Anthozoa, Cubozoa, Hydrozoa, Polypodiozoa, Scyphozoa, Staurozoa, and Myxozoa. Myxozoa comprises two subclasses of obligate parasites—Myxosporea and Malacosporea, which demonstrate various degrees of simplification. Myxosporea were previously reported to lack the majority of core protein domains of apoptotic proteins including caspases, Bcl-2, and APAF-1 homologs. Other sequenced Cnidaria, including the parasite Polypodium hydriforme from Polypodiozoa do not share this genetic feature. Whether this loss of core apoptotic proteins is unique to Myxosporea or also present in its sister subclass Malacosporea was not previously investigated. We show that the presence of core apoptotic proteins gradually diminishes from free-living Cnidaria to Polypodium to Malacosporea to Myxosporea. This observation does not favor the hypothesis of catastrophic simplification of Myxosporea at the genetic level, but rather supports a stepwise adaptation to parasitism that likely started from early parasitic ancestors that gave rise to Myxozoa.

Innate Conformational Dynamics Drive Binding Specificity in Anti-Apoptotic Proteins Mcl-1 and Bcl-2.

The structurally conserved B-cell lymphoma 2 (Bcl-2) family of protein function to promote or inhibit apoptosis through an exceedingly complex web of specific, intrafamilial protein-protein interactions. The critical role of these proteins in lymphomas and other cancers has motivated a widespread interest in understanding the molecular mechanisms that drive specificity in Bcl-2 family interactions. However, the high degree of structural similarity among Bcl-2 homologues has made it difficult to rationalize the highly specific (and often divergent) binding behavior exhibited by these proteins using conventional structural arguments. In this work, we use time-resolved hydrogen deuterium exchange mass spectrometry to explore shifts in conformational dynamics associated with binding partner engagement in the Bcl-2 family proteins Bcl-2 and Mcl-1. Using this approach combined with homology modeling, we reveal that Mcl-1 binding is driven by a large-scale shift in conformational dynamics, while Bcl-2 complexation occurs primarily through a classical charge compensation mechanism. This work has implications for understanding the evolution of internally regulated biological systems composed of structurally similar proteins and for the development of drugs targeting Bcl-2 family proteins for promotion of apoptosis in cancer.

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a β-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

Abstract LB304: Mechanisms of cell death escape in glioblastoma

Abstract Glioblastoma is the most common brain tumor in adults and among the deadliest malignancies per se with a highly invasive phenotype upon presentation. To achieve rapid colonialization throughout the central nervous system, glioblastoma cells have to be equipped with a high resistance to several forms of programmed cell death, such as apoptosis. All this occurs in the absence of any tumor-initiated signature mutations. Using a comprehensive comparative analysis combining expression profiles and functional analysis of normal brain glia cells, primary tumors and tumor-derived organoids with distinct differentiation subtypes we investigated the underlying features associated with the high resistance to cell death-induced by conventional treatment. To break this resistance, we looked into the possibility to add the small molecule inhibitor Venetoclax, that targets the Bcl-2 family, to conventional therapy. The Bcl-2 family are a number of evolutionarily-conserved proteins that share Bcl-2 homology (BH) domains and are most notable for their regulation of apoptosis at the mitochondrion. Interestingly, although stem cell-like cells (SCs) express more pro-apoptotic Bcl-2 family proteins than their differentiated progeny they remain more resistant to apoptosis, suggesting that the Bcl-2 family is not the main mediator of apoptosis resistance. In stark contrast to leukemia cells, inhibition of Bcl-2 alone has no effect on glioblastoma cells, but combining this with either Temozolomide (TMZ), the standard chemotherapeutic option, or serum starvation leads to synergistic effects. These are, however, weak hence we investigated whether compensatory mechanisms are activated. Indeed, in both SCs and DCs we found an upregulation of Mcl-1, a molecule known to compensate for inhibited Bcl-2. However, additionally blocking Mcl-1 with several different small molecule inhibitors did not further sensitize primary Glioblastoma cells. This suggests that while mechanical upregulation of Mcl-1 occurs in Glioblastoma, it is of little functional consequence. The reduced importance of the Bcl-2 family is also reflected in the non-transformed astrocytes which are precursor of glioblastoma. They already display an intrinsic high resistance to apoptotic cell death. Treatment with TMZ and modulators of apoptosis does not significantly affect their viability. Our findings suggest that the Bcl-2 family has a reduced role in mediating the survival of brain cells, at least when compared to haemopoietic cells. While a certain sensitizing effect can be achieved in Glioblastoma cells by combining cellular stressors with inhibitors of the Bcl-2 family, this is unlikely to be a sufficient to overcome therapy resistance. Citation Format: Helene von Bandemer, Felix Seyfried, Anne Fleischmann, Georg Karpel-Massler, Aurelia Peraud, Hannah Strobel, Marcin Lyszkiewicz, Klaus Michael Debatin, Mike Andrew Westhoff. Mechanisms of cell death escape in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB304.

Mitochondrial Bcl-xL promotes brain synaptogenesis by controlling non-lethal caspase activation

Non-lethal caspase activation (NLCA) has been linked to neurodevelopmental processes. However, how neurons control NLCA remains elusive. Here, we focused on Bcl-xL, a Bcl-2 homolog regulating caspase activation through the mitochondria. We generated a mouse model, referred to as ER-xL, in which Bcl-xL is absent in the mitochondria, yet present in the endoplasmic reticulum. Unlike bclx knockout mice that died at E13.5, ER-xL mice survived embryonic development but died post-partum because of altered feeding behavior. Enhanced caspase-3 activity was observed in the brain and the spinal cord white matter, but not the gray matter. No increase in cell death was observed in ER-xL cortical neurons, suggesting that the observed caspase-3 activation was apoptosis-independent. ER-xL neurons displayed increased caspase-3 activity in the neurites, resulting in impaired axon arborescence and synaptogenesis. Together, our findings suggest that mitochondrial Bcl-xL finely tunes caspase-3 through Drp-1-dependent mitochondrial fission, which is critical to neural network design.

A crosstalk between phosphorylation and ubiquitination of BNIP3 regulates mitophagy under hypoxia

BNIP3 (BCL2/adenovirus e1B 19 kDa protein interacting protein 3) is a mitochondrial outer membrane protein that is sensitive to hypoxia and mediates mitophagy, a process important for mitochondrial quality control and to maintain energetic and redox homeostasis under hypoxia. It has been reported that up-regulation of BNIP3, which acts as mitophagy receptor, promotes mitophagy. In our recent study, we found that the post-translational modification of BNIP3 is crucial to induce mitophagy, and that a crosstalk between phosphorylation/dephosphorylation and ubiquitination acts as a switch to control BNIP3-mediated mitophagy under hypoxia. We demonstrated that the phosphorylation of BNIP3 at S60 and T66 by MAPK8/9 (mitogen-activated protein kinase 8/9) under hypoxia blocks the degradation of BNIP3 via the ubiquitin-proteasome pathway and enhances its interaction with MAP1LC3 (microtubule associated protein 1 light chain 3), thereby promoting mitophagy. In contrast, dephosphorylation of BNIP3 by members of the PP1/2A (protein phosphatase PP1 and PP2A) phosphatase subfamily under hypoxia accelerates degradation of BNIP3 via the ubiquitin-proteasome pathway, thereby suppressing mitophagy. Altogether, these findings provide knowledge necessary to devise intervention strategies for hypoxia-related diseases and/or hypoxia-related developmental processes.Abbreviations: BCL2: BCL2 apoptosis regulator; BCL2L1: BCL2 like 1; BECN1: beclin 1, autophagy related; BH3: BCL2 homology 3; BNIP3: BCL2/adenovirus e1B 19 kDa protein interacting protein 3; LIR: MAP1LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; MAPK8: mitogen-activated protein kinase 8; MAPK9: mitogen-activated protein kinase 9; PEST: rich in amino acids P, E, S, T, and D; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; PEST: rich in amino acids P, E, S, T, and D

Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process.

B-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned, and its role in apoptosis was investigated. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. In vivo overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- κB (NF-κB) signaling pathway-related genes in vitro were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-κB signaling pathway. The full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant "NWGR" motif located in BH1 were identified in TroBcl2. In healthy T. ovatus, TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as BOK, caspase-9, caspase-7, caspase-3, cytochrome c, and p53. Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-κB and regulated the expression of genes (such as NF-κB1 and c-Rel) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1β. Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in T. ovatus.

Identification of Potential Genes and Critical Pathways in Postoperative Recurrence of Crohn's Disease by Machine Learning And WGCNA Network Analysis.

Crohn's disease (CD) is a chronic idiopathic inflammatory bowel disease affecting the entire gastrointestinal tract from the mouth to the anus. These patients often experience a period of symptomatic relapse and remission. A 20 - 30% symptomatic recurrence rate is reported in the first year after surgery, with a 10% increase each subsequent year. Thus, surgery is done only to relieve symptoms and not for the complete cure of the disease. The determinants and the genetic factors of this disease recurrence are also not well-defined. Therefore, enhanced diagnostic efficiency and prognostic outcome are critical for confronting CD recurrence. We analysed ileal mucosa samples collected from neo-terminal ileum six months after surgery (M6=121 samples) from Crohn's disease dataset (GSE186582). The primary aim of this study is to identify the potential genes and critical pathways in post-operative recurrence of Crohn's disease. We combined the differential gene expression analysis with Recursive feature elimination (RFE), a machine learning approach to get five critical genes for the postoperative recurrence of Crohn's disease. The features (genes) selected by different methods were validated using five binary classifiers for recurrence and remission samples: Logistic Regression (LR), Decision tree classifier (DT), Support Vector Machine (SVM), Random Forest classifier (RF), and K-nearest neighbor (KNN) with 10-fold cross-validation. We also performed weighted gene co-expression network analysis (WGCNA) to select specific modules and feature genes associated with Crohn's disease postoperative recurrence, smoking, and biological sex. Combined with other biological interpretations, including Gene Ontology (GO) analysis, pathway enrichment, and protein-protein interaction (PPI) network analysis, our current study sheds light on the in-depth research of CD diagnosis and prognosis in postoperative recurrence. PLOD2, ZNF165, BOK, CX3CR1, and ARMCX4, are the important genes identified from the machine learning approach. These genes are reported to be involved in the viral protein interaction with cytokine and cytokine receptors, lysine degradation, and apoptosis. They are also linked with various cellular and molecular functions such as Peptidyl-lysine hydroxylation, Central nervous system maturation, G protein-coupled chemoattractant receptor activity, BCL-2 homology (BH) domain binding, Gliogenesis and negative regulation of mitochondrial depolarization. WGCNA identified a gene co-expression module that was primarily involved in mitochondrial translational elongation, mitochondrial translational termination, mitochondrial translation, mitochondrial respiratory chain complex, mRNA splicing via spliceosome pathways, etc.; Both the analysis result emphasizes that the mitochondrial depolarization pathway is linked with CD recurrence leading to oxidative stress in promoting inflammation in CD patients. These key genes serve as the novel diagnostic biomarker for the postoperative recurrence of Crohn's disease. Thus, among other treatment options present until now, these biomarkers would provide success in both diagnosis and prognosis, aiming for a long-lasting remission to prevent further complications in CD.

Bcl-2 expression stimulates ethanol production in yeast.

It is now well established that shifts in energy metabolism are associated with cancer development and progression. The most studied of these phenomena is the Warburg effect, which corresponds to an increase of anaerobic glycolysis vs mitochondrial oxidative phosphorylation to produce energy for cellular processes. However, the mechanisms related to these metabolic switches are still a matter of debate. Bcl-2 family proteins contain both pro- (e.g. Bax), and anti-apoptotic (e.g. Bcl-2 and Bcl-xL) members which are respectively encoded by tumor suppressors and proto-oncogenes. Up-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL have been associated with Non-Hodgkin's lymphoma; and certain studies indicate that Bcl-2 family proteins play a role in the regulation of energy metabolism. However, the definition of the molecular mechanisms involved in Bcl-2/xL regulation of carbohydrate metabolism are challenging to define as mammalian cell models contain several Bcl-2/xL homologs with possible redundant functions. The yeast S. cerevisiae is a eukaryotic cell model which possesses the advantages of: (i) to undergo natural carbohydrate metabolic shifts; and (ii) not to possess any Bcl-2 family homologs. A 13C-NMR based detection and quantification of extracellular glucose-derived metabolites allowed us to recently observe that Bcl-2 expression was associated with a significant increase of alcoholic fermentation in yeast. These data suggest that Bcl-2 expression levels may play an active role in the stimulation of lactic fermentation commonly observed in blood cancer cells. These data also suggest that this stimulation of fermentation by Bcl-2 is direct; and that it does not require the support of other Bcl-2 homologs.

Phylogenetic analysis of the MCL1 BH3 binding groove and rBH3 sequence motifs in the p53 and INK4 protein families.

B-cell lymphoma 2 (Bcl-2) proteins are central, conserved regulators of apoptosis. Bcl-2 family function is regulated by binding interactions between the Bcl-2 homology 3 (BH3) motif in pro-apoptotic family members and the BH3 binding groove found in both the pro-apoptotic effector and anti-apoptotic Bcl-2 family members. A novel motif, the reverse BH3 (rBH3), has been shown to interact with the anti-apoptotic Bcl-2 homolog MCL1 (Myeloid cell leukemia 1) and have been identified in the p53 homolog p73, and the CDK4/6 (cyclin dependent kinase 4/6) inhibitor p18INK4c, (p18, cyclin-dependent kinase 4 inhibitor c). To determine the conservation of rBH3 motif, we first assessed conservation of MCL1's BH3 binding groove, where the motif binds. We then constructed neighbor-joining phylogenetic trees of the INK4 and p53 protein families and analyzed sequence conservation using sequence logos of the rBH3 locus. This showed the rBH3 motif is conserved throughout jawed vertebrates p63 and p73 sequences and in chondrichthyans, amphibians, mammals, and some reptiles in p18. Finally, a potential rBH3 motif was identified in mammalian and osteichthyan p19INK4d (p19, cyclin dependent kinase 4 inhibitor d). These findings demonstrate that the interaction between MCL1 and other cellular proteins mediated by the rBH3 motif may be conserved throughout jawed vertebrates.

Endoplasmic reticulum protein BIK binds to and inhibits mitochondria-localized antiapoptotic proteins

The proapoptotic BCL-2 homology (BH3)-only endoplasmic reticulum (ER)-resident protein BCL-2 interacting killer (BIK) positively regulates mitochondrial outer membrane permeabilization, the point of no return in apoptosis. It is generally accepted that BIK functions at a distance from mitochondria by binding and sequestering antiapoptotic proteins at the ER, thereby promoting ER calcium release. Although BIK is predominantly localized to the ER, we detect by fluorescence lifetime imaging microscopy-FRET microscopy, BH3 region-dependent direct binding between BIK and mitochondria-localized chimeric mutants of the antiapoptotic proteins BCL-XL and BCL-2 in both baby mouse kidney (BMK) and MCF-7cells. Direct binding was accompanied by cell type-specific differential relocalization in response to coexpression of either BIK or one of its target binding partners, BCL-XL, when coexpressed in cells. In BMK cells with genetic deletion of both BAX and BAK (BMK-double KO), our data suggest that a fraction of BIK protein moves toward mitochondria in response to the expression of a mitochondria-localized BCL-XL mutant. In contrast, in MCF-7cells, our data suggest that BIK is localized at both ER and mitochondria-associated ER membranes and binds to the mitochondria-localized BCL-XL mutant via relocalization of BCL-XL to ER and mitochondria-associated ER membrane. Rather than functioning at a distance, our data suggest that BIK initiates mitochondrial outer membrane permeabilization via direct interactions with ER and mitochondria-localized antiapoptotic proteins, which occur via ER-mitochondria contact sites, and/or by relocalization of either BIK or antiapoptotic proteins in cells.

Structural analysis of the interaction between Bcl-xL and the noncanonical BH3 domain of non-Bcl-2 family proteins.

Anti-apoptotic and anti-autophagic Bcl-2 homologues commonly contain a hydrophobic groove in which the BH3 domain is accommodated. The BH3 domain is usually considered a feature of Bcl-2 family members; however, it has also been found in various non-Bcl-2 family proteins. Although interactions among Bcl-2 family members have been extensively investigated and highlighted, those mediated by the BH3 domain of non-Bcl-2 family proteins have not been the focus of substantial research. In this review, the author conducted a structural analysis of Bcl-xL complexed with the BH3 domain of four non-Bcl-2 family proteins, Beclin 1, SOUL, TCTP, and Pxt1, at an atomic level. Although the overall Bcl-xL-binding modes are similar among these proteins, they are characterized by limited sequence conservation of the BH3 consensus motif and differences in residues involved in complex formation. Based on the structural analysis, the authorsuggests that more "undiscovered" BH3 domain-containing proteins might exist, which have been unidentified due to their limited sequence conservation but can bind to Bcl-2 family proteins and control apoptosis, autophagy, or other biological processes.

Biological properties of the BCL-2 family protein BCL-RAMBO, which regulates apoptosis, mitochondrial fragmentation, and mitophagy.

Mitochondria play an essential role in the regulation of cellular stress responses, including cell death. Damaged mitochondria are removed by fission and fusion cycles and mitophagy, which counteract cell death. BCL-2 family proteins possess one to four BCL-2 homology domains and regulate apoptosis signaling at mitochondria. BCL-RAMBO, also known as BCL2-like 13 (BCL2L13), was initially identified as one of the BCL-2 family proteins inducing apoptosis. Mitophagy receptors recruit the ATG8 family proteins MAP1LC3/GABARAP via the MAP1LC3-interacting region (LIR) motif to initiate mitophagy. In addition to apoptosis, BCL-RAMBO has recently been identified as a mitophagy receptor that possesses the LIR motif and regulates mitochondrial fragmentation and mitophagy. In the 20years since its discovery, many important findings on BCL-RAMBO have been increasingly reported. The biological properties of BCL-RAMBO are reviewed herein.

Repurposing BCL-2 and Jak 1/2 inhibitors: Cure and treatment of HIV-1 and other viral infections.

B cell lymphoma 2 (BCL-2) family proteins are involved in the mitochondrial apoptotic pathway and are key modulators of cellular lifespan, which is dysregulated during human immunodeficiency virus type 1 (HIV-1) and other viral infections, thereby increasing the lifespan of cells harboring virus, including the latent HIV-1 reservoir. Long-lived cells harboring integrated HIV-1 DNA is a major barrier to eradication. Strategies reducing the lifespan of reservoir cells could significantly impact the field of cure research, while also providing insight into immunomodulatory strategies that can crosstalk to other viral infections. Venetoclax is a first-in-class orally bioavailable BCL-2 homology 3 (BH3) mimetic that recently received Food and Drug Administration (FDA) approval for treatment in myeloid and lymphocytic leukemia. Venetoclax has been recently investigated in HIV-1 and demonstrated anti-HIV-1 effects including a reduction in reservoir size. Another immunomodulatory strategy towards reduction in the lifespan of the reservoir is Jak 1/2 inhibition. The Jak STAT pathway has been implicated in BCL-2 and interleukin 10 (IL-10) expression, leading to a downstream effect of cellular senescence. Ruxolitinib and baricitinib are FDA-approved, orally bioavailable Jak 1/2 inhibitors that have been shown to indirectly decay the HIV-1 latent reservoir, and down-regulate markers of HIV-1 persistence, immune dysregulation and reservoir lifespan in vitro and ex vivo. Ruxolitinib recently demonstrated a significant decrease in BCL-2 expression in a human study of virally suppressed people living with HIV (PWH), and baricitinib recently received emergency use approval for the indication of coronavirus disease 2019 (COVID-19), underscoring their safety and efficacy in the viral infection setting. BCL-2 and Jak 1/2 inhibitors could be repurposed as immunomodulators for not only HIV-1 and COVID-19, but other viruses that upregulate BCL-2 anti-apoptotic proteins. This review examines potential routes for BCL-2 and Jak 1/2 inhibitors as immunomodulators for treatment and cure of HIV-1 and other viral infections.

Sensitivity of canine hematological cancers to BH3 mimetics.

Inhibition of antiapoptotic B-cell lymphoma 2 (BCL2) proteins by small molecule Bcl-2 homology 3 (BH3) mimetics causes rapid induction of apoptosis of human hematological cancers in vitro and in vivo. Assess in vitro sensitivity of non-neoplastic lymphocytes and primary hematological cancer cells from dogs to venetoclax (VEN) or the dual BCL2/ B-cell lymphoma-extra-large (BCLxL) inhibitor, navitoclax (NAV), and evaluate the association between BCL2 protein expression and VEN sensitivity. Nine client-owned dogs without cancer and 18 client-owned dogs with hematological cancer. Prospective, nonrandomized noncontrolled study. Lymphocytes isolated from peripheral blood, lymph node, or bone marrow from dogs were incubated with BH3 mimetics for 24 hours. Viable cells were counted using flow cytometry and half maximal effective concentration (EC50 ) was calculated. BCL2 protein from whole cell lysates was assessed via immunoblots. Nodal B and T lymphocytes were more sensitive to VEN than circulating lymphocytes (P=.02). Neoplastic T lymphocytes were sensitive to VEN (mean EC50 ± SD =0.023 ± 0.018 μM), whereas most non-indolent B cell cancers were resistant to killing by VEN (mean EC50 ± SD=288 ± 700 μM). Unclassified leukemias showed variable sensitivity to VEN (mean EC50 ± SD=0.49 ± 0.66 μM). Detection of BCL2 protein was not associated with VEN sensitivity. Neoplastic canine T lymphocytes are sensitive to VEN in vitro. Quantification of BCL2 protein alone is insufficient to predict sensitivity to VEN.

Expression Ratios of the Antiapoptotic BCL2 Family Members Dictate the Selective Addiction of Kaposi's Sarcoma-Associated Herpesvirus-Transformed Primary Effusion Lymphoma Cell Lines to MCL1.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people living with HIV, including primary effusion lymphoma (PEL). PEL cell lines exhibit oncogene addictions to both viral and cellular genes. Using CRISPR screens, we previously identified cellular oncogene addictions in PEL cell lines, including MCL1. MCL1 is a member of the BCL2 family, which functions to prevent intrinsic apoptosis and has been implicated in several cancers. Despite the overlapping functions of the BCL2 family members, PEL cells are dependent only on MCL1, suggesting that MCL1 may have nonredundant functions. To investigate why PEL cells exhibit selective addiction to MCL1, we inactivated the intrinsic apoptosis pathway by engineering BAX/BAK1 double knockout cells. In this context, PEL cells become resistant to MCL1 knockdown or MCL1 inactivation by the MCL1 inhibitor S63845, indicating that the main function of MCL1 in PEL cells is to prevent BAX/BAK1-mediated apoptosis. The selective requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Ectopic expression of several BCL2 family proteins, as well as the KSHV BCL2 homolog, significantly decreased basal caspase 3/7 activity and buffered against staurosporine-induced apoptosis. Finally, overexpressed BCL2 family members can functionally substitute for MCL1, when it is inhibited by S63845. Together, our data indicate that the expression levels of the BCL2 family likely explain why PEL tumor cells are highly addicted to MCL1. Importantly, our results suggest that caution should be taken when considering MCL1 inhibitors as a monotherapy regimen for PEL because resistance can develop easily. IMPORTANCE Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus. We showed previously that PEL cell lines require the antiapoptotic protein MCL1 for survival but not the other BCL2 family proteins. This selective dependence on MCL1 is unexpected as the BCL2 family functions similarly in preventing intrinsic apoptosis. Recently, new roles for MCL1 not shared with the BCL2 family have emerged. Here, we show that noncanonical functions of MCL1 are unlikely essential. Instead, MCL1 functions mainly to prevent apoptosis. The specific requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Consistent with this model, shifting these expression ratios changes the requirement away from MCL1 and toward the dominant BCL2 family gene. Together, our results indicate that although MCL1 is an attractive chemotherapeutic target to treat PEL, careful consideration must be taken, as resistance to MCL1-specific inhibitors easily develops through BCL2 family overexpression.

Development of a Robust BH3 Profiling Toolkit for Precision Medicine in Hematologic Malignancies

INTRODUCTION. BCL2 homology 3 (BH3) mimetics represent a novel class of pro-apoptotic anti Hematologic malignancies (HM) drugs directly targeting apoptosis, even in chemoresistant cells. Each disease is heterogenous which makes it hard to provide standardized treatment for each patient and highlight the need for new diagnosis tools to deliver precision medicine. In the other hand, a technic called BH3 profiling has been developed, allowing to measure the apoptosis priming of the cells as well as their dependencies on specific anti-apoptotic proteins. Using specific BH3 peptides, this assay measures the percentage of cytochrome c release for each anti-apoptotic protein, mainly Bcl2, Mcl1 and Bcl-xl. BH3 toolkit, a variation of this technic, was developed using BH3 mimetics instead of BH3 peptides. Those small molecules mimic the effect of BH3 sensitizer protein by competing for anti-apoptotic protein catalytic pocket. Using these drugs allows to avoid cell permeabilization and thus reduce variability in the assay. A dynamic BH3 toolkit is also possible, measuring early drug-induced changes in net proapoptotic signaling, to identify modification of antiapoptotic dependencies. The aim of the present work is to develop a standardize and reproducible BH3 toolkit (baseline and dynamic), with internal control, to allow an absolute measurement of specific anti-apoptotic dependencies in HM cell lines or primary tumor cells, as well as identifying optimal therapeutic combination. MATERIAL AND METHODS. We used different HM cell lines as well as primary cells from patients with CLL. Cells were seeded right after thawing at the concentration of 200,000 cells/mL in a 96 well plate, using a robotic liquid handler. Primary cells were cocultured with stromal cells (Resto-6 cell line). Cells are then incubated for 20h in a 5% CO2 and 37°C incubator. Next, we treated them with BH3 mimetics (or vehicle): Venetoclax (VEN, Bcl2 inhibitor), Navitoclax (Bcl2 and Bcl-xl inhibitor), AZD-5991 (AZD, Mcl1 inhibitor), A-1155463 (Bcl-xl inhibitor) for 4h. Then, cells were stained using Annexin V and 7AAD and analyzed by flow cytometry. Our gating strategy focuses on live cells to measure annexin V flipflop as an apoptotic marker. This assay is paired with a cell viability measurement at 24h, using CellTiterGlo assay. For dynamic BH3 toolkit, the influence of the tested drug on antiapoptotic dependencies is measured by adding this drug (or vehicle) at the same time as cells of interest. RESULTS. First, we wanted to optimize the protocol on cell lines with known anti-apoptotic dependencies. We used OCI-Ly1 and LP1 cell lines which are known for their exclusive Bcl2 and Mcl1 dependencies, respectively. As expected, OCI-Ly1 cells showed a significant increase in annexin V staining (reaching 48.2% (P=0.0003) at maximum concentration) upon VEN treatment, whereas it does not change significantly with Mcl1 or Bcl-xl inhibitors (Figure A). Likewise, we detected an increase of 15.35% (P=0.005) in Annexin V staining only upon AZD in LP1 cells, but no significant modification with other inhibitors. Consistent with these data, we saw a significant and specific decrease in cell viability (CTG) with VEN and AZD on OCI-Ly1 and LP1 respectively (data no shown). These 2 cell lines are used as internal controls for the other analyses. Next, we optimized the assay on CLL primary cells. Figure A shows that VEN induces an increase in Annexin V staining by 62.7 % (P=0.0003), demonstrating a significant Bcl2 dependency. Finally, we optimized the dynamic part of the assay (Figure B). We used ironomycin, a lysosomal iron-targeting small molecule that has been studied by our team in HM (Devin et al. 2022). We found that ironomycin increased significantly Bcl2 dependency in OCI-Ly1 cells by 8.55% (P=0.02) with VEN. Interestingly, we observed the induction of a MCL1 dependency in those cells, with an annexin V staining increase by 8.21% (P=0.02). CONCLUSION. We were therefore able to measure the baseline dependencies using a BH3 toolkit that avoid cell permeabilization. We were also able to measure changes in dependencies induced by an innovative therapy. We believe that our results show the robustness of the BH3 toolkit and its ability to detect potential new therapeutic strategies in HM cell lines and primary cells. Detailed results will be presented at the meeting, as well as data on primary lymphoma and myeloma cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal