Human parainfluenza virus type 2 V protein inhibits mitochondrial apoptosis pathway through two ways.

Paramyxoviruses are reported to block apoptosis for their replication, but the mechanisms remain unclear. Furthermore, regulation of mitochondrial apoptosis by paramyxoviruses has been hardly reported. We investigated whether and how human parainfluenza virus type 2 (hPIV-2) counteracts apoptosis. Infection of recombinant hPIV-2 carrying mutated V protein showed higher caspase 3/7 activity and higher cytochrome c release from mitochondria than wild type hPIV-2 infection. This indicates that V protein controls mitochondrial apoptosis pathway. hPIV-2 V protein interacted with Bad, an apoptotic promoting protein, and this interaction inhibited the binding of Bad to Bcl-XL. V protein also bound to 14-3-3ε, which was essential for inhibition of 14-3-3ε cleavage. Our data collectively suggest that hPIV-2 V protein has two means of preventing mitochondrial apoptosis pathway: the inhibition of Bad-Bcl-XL interaction and the suppression of 14-3-3ε cleavage. This is the first report of the mechanisms behind how paramyxoviruses modulate mitochondrial apoptosis pathways.

PROTAC-Mediated Dual Degradation of BCL-xL and BCL-2 Is a Highly Effective Therapeutic Strategy in Small-Cell Lung Cancer.

BCL-xL and BCL-2 are validated therapeutic targets in small-cell lung cancer (SCLC). Targeting these proteins with navitoclax (formerly ABT263, a dual BCL-xL/2 inhibitor) induces dose-limiting thrombocytopenia through on-target BCL-xL inhibition in platelets. Therefore, platelet toxicity poses a barrier in advancing the clinical translation of navitoclax. We have developed a strategy to selectively target BCL-xL in tumors, while sparing platelets, by utilizing proteolysis-targeting chimeras (PROTACs) that hijack the cellular ubiquitin proteasome system for target ubiquitination and subsequent degradation. In our previous study, the first-in-class BCL-xL PROTAC, called DT2216, was shown to have synergistic antitumor activities when combined with venetoclax (formerly ABT199, BCL-2-selective inhibitor) in a BCL-xL/2 co-dependent SCLC cell line, NCI-H146 (hereafter referred to as H146), in vitro and in a xenograft model. Guided by these findings, we evaluated our newly developed BCL-xL/2 dual degrader, called 753b, in three BCL-xL/2 co-dependent SCLC cell lines and the H146 xenograft models. 753b was found to degrade both BCL-xL and BCL-2 in these cell lines. Importantly, it was considerably more potent than DT2216, navitoclax, or DT2216 + venetoclax in reducing the viability of BCL-xL/2 co-dependent SCLC cell lines in cell culture. In vivo, 5 mg/kg weekly dosing of 753b was found to lead to significant tumor growth delay, similar to the DT2216 + venetoclax combination in H146 xenografts, by degrading both BCL-xL and BCL-2. Additionally, 753b administration at 5 mg/kg every four days induced tumor regressions. At this dosage, 753b was well tolerated in mice, without observable induction of severe thrombocytopenia as seen with navitoclax, and no evidence of significant changes in mouse body weights. These results suggest that the BCL-xL/2 dual degrader could be an effective and safe therapeutic for a subset of SCLC patients, warranting clinical trials in future.

In silico design of potential Mcl-1 peptide-based inhibitors.

CONTEXT: BIM (Bcl-2 interacting mediator of apoptosis)-derived peptides that specifically target over-expressed Mcl-1 (myeloid cell leukemia-1) protein and induce apoptosis are potentially anti-cancer agents. Since the helicity of BIM-derived peptides has a crucial role in their functionality, a range of strategies have been used to increase the helicity including the introduction of unnatural residues and stapling methods that have some drawbacks such as the accumulation in the liver. To avoid these drawbacks, this study aimed to design a more helical peptide by utilizing bioinformatics algorithms and molecular dynamics simulations without exploiting unnatural residues and stapling methods. MM-PBSA results showed that the mutations of A4fE and A2eE in analogue 5 demonstrate a preference towards binding with Mcl-1. As evidenced by Circular dichroism results, the helicity increases from 18 to 34%, these findings could enhance the potential of analogue 5 as an anti-cancer agent targeting Mcl-1. The applied strategies in this research could shed light on the in silico peptide design. Moreover, analogue 5 as a drug candidate can be evaluated in vitro and in vivo studies. METHODS: The sequence of the lead peptide was determined using the ApInAPDB database and PRALINE program. Contact finder and PDBsum web server softwares were used to determine the contact involved amino acids in complex with Mcl-1. All identified salt bridge contributing residues were unaltered to preserve the binding affinity. After proposing novel analogues, their secondary structures were predicted by Cham finder web server software and GOR, Neural Network, and Chou-Fasman algorithms. Finally, molecular dynamics simulations run for 100 ns were done using the GROMACS, version 5.0.7, with the CHARMM36 force field. MM-PBSA was used to assess binding affinity specificity in targeting Mcl-1 and Bcl-xL (B-cell lymphoma extra-large).

Development and crystal structures of a potent second-generation dual degrader of BCL-2 and BCL-xL.

Overexpression of BCL-xL and BCL-2 play key roles in tumorigenesis and cancer drug resistance. Advances in PROTAC technology facilitated recent development of the first BCL-xL/BCL-2 dual degrader, 753b, a VHL-based degrader with improved potency and reduced toxicity compared to previous small molecule inhibitors. Here, we determine crystal structures of VHL/753b/BCL-xL and VHL/753b/BCL-2 ternary complexes. The two ternary complexes exhibit markedly different architectures that are accompanied by distinct networks of interactions at the VHL/753b-linker/target interfaces. The importance of these interfacial contacts is validated via functional analysis and informed subsequent rational and structure-guided design focused on the 753b linker and BCL-2/BCL-xL warhead. This results in the design of a degrader, WH244, with enhanced potency to degrade BCL-xL/BCL-2 in cells. Using biophysical assays followed by in cell activities, we are able to explain the enhanced target degradation of BCL-xL/BCL-2 in cells. Most PROTACs are empirically designed and lack structural studies, making it challenging to understand their modes of action and specificity. Our work presents a streamlined approach that combines rational design and structure-based insights backed with cell-based studies to develop effective PROTAC-based cancer therapeutics.

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.

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer.

Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

Cyclic peptides discriminate BCL-2 and its clinical mutants from BCL-XL by engaging a single-residue discrepancy.

Overexpressed pro-survival B-cell lymphoma-2 (BCL-2) family proteins BCL-2 and BCL-XL can render tumor cells malignant. Leukemia drug venetoclax is currently the only approved selective BCL-2 inhibitor. However, its application has led to an emergence of resistant mutations, calling for drugs with an innovative mechanism of action. Herein we present cyclic peptides (CPs) with nanomolar-level binding affinities to BCL-2 or BCL-XL, and further reveal the structural and functional mechanisms of how these CPs target two proteins in a fashion that is remarkably different from traditional small-molecule inhibitors. In addition, these CPs can bind to the venetoclax-resistant clinical BCL-2 mutants with similar affinities as to the wild-type protein. Furthermore, we identify a single-residue discrepancy between BCL-2 D111 and BCL-XL A104 as a molecular "switch" that can differently engage CPs. Our study suggests that CPs may inhibit BCL-2 or BCL-XL by delicately modulating protein-protein interactions, potentially benefiting the development of next-generation therapeutics.

Computational Insights into the Interaction of Pinostrobin with Bcl-2 Family Proteins: A Molecular Docking Analysis.

BACKGROUND: Cancer research has emphasized the Bcl-2 family of proteins because of their interaction in apoptosis process, a critical mechanism that regulates cellular survival and death. Recently small molecules from diverse sources have gained much attention in anticancer research due to their promising inhibitory action against Bcl-2 and Bcl-XL that are pointedly known as the members of anti-apoptotic Bcl-2 family of proteins. Pinostrobin (PN) is a natural flavonoid with diverse pharmacological potential emerged as a molecule of interest as anticancer agent. The present study aims to screen the interaction of PN with anti-apoptotic protagonists Bcl-2 and Bcl- XL at the molecular level through docking studies. METHOD: The molecular docking was performed using the Schrodinger software. The docking score of PN with the Bcl-2 (4IEH) and Bcl-XL (3ZK6) and their molecular interactions was examined and analysed. RESULTS: The result of the molecular docking analysis showed that PN and the anti-apoptotic proteins 4IEH and 3ZK6 had significant interactions and docking energy scores (ΔG) were found to be -5.112 kcal/mol and -7.822 kcal/mol respectively. The small molecule PN illustrated effective interaction with the active site amino acids of the Bcl-2 and Bcl-XL proteins and has been associated through traditional hydrogen bond with 4IEH. Further, it was observed that PN and anti-apoptotic Bcl-2 proteins interaction was stabilized by other non-covalent interactions, such as π-alkyl or π-π interactions and van der Waals forces. CONCLUSIONS: This was the first study to reveal the inhibitory action of PN against anti-apoptotic Bcl-2 and Bcl-XL proteins at the molecular level. The findings of this study concludes that PN ability to inhibit anti-apoptotic proteins, Bcl-2 and Bcl-XL could be useful to induce intracellular apoptosis in tumorous cells.

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

Genome-wide CRISPR screen reveals the synthetic lethality between BCL2L1 inhibition and radiotherapy.

Radiation therapy (RT) is one of the most commonly used anticancer therapies. However, the landscape of cellular response to irradiation, especially to a single high-dose irradiation, remains largely unknown. In this study, we performed a whole-genome CRISPR loss-of-function screen and revealed temporal inherent and acquired responses to RT. Specifically, we found that loss of the IL1R1 pathway led to cellular resistance to RT. This is in part because of the involvement of radiation-induced IL1R1-dependent transcriptional regulation, which relies on the NF-κB pathway. Moreover, the mitochondrial anti-apoptotic pathway, particularly the BCL2L1 gene, is crucially important for cell survival after radiation. BCL2L1 inhibition combined with RT dramatically impeded tumor growth in several breast cancer cell lines and syngeneic models. Taken together, our results suggest that the combination of an apoptosis inhibitor such as a BCL2L1 inhibitor with RT may represent a promising anticancer strategy for solid cancers including breast cancer.

Modification of BCLX pre-mRNA splicing has antitumor efficacy alone or in combination with radiotherapy in human glioblastoma cells.

Dysregulation of anti-apoptotic and pro-apoptotic protein isoforms arising from aberrant splicing is a crucial hallmark of cancers and may contribute to therapeutic resistance. Thus, targeting RNA splicing to redirect isoform expression of apoptosis-related genes could lead to promising anti-cancer phenotypes. Glioblastoma (GBM) is the most common type of malignant brain tumor in adults. In this study, through RT-PCR and Western Blot analysis, we found that BCLX pre-mRNA is aberrantly spliced in GBM cells with a favored splicing of anti-apoptotic Bcl-xL. Modulation of BCLX pre-mRNA splicing using splice-switching oligonucleotides (SSOs) efficiently elevated the pro-apoptotic isoform Bcl-xS at the expense of the anti-apoptotic Bcl-xL. Induction of Bcl-xS by SSOs activated apoptosis and autophagy in GBM cells. In addition, we found that ionizing radiation could also modulate the alternative splicing of BCLX. In contrast to heavy (carbon) ion irradiation, low energy X-ray radiation-induced an increased ratio of Bcl-xL/Bcl-xS. Inhibiting Bcl-xL through splicing regulation can significantly enhance the radiation sensitivity of 2D and 3D GBM cells. These results suggested that manipulation of BCLX pre-mRNA alternative splicing by splice-switching oligonucleotides is a novel approach to inhibit glioblastoma tumorigenesis alone or in combination with radiotherapy.

Gpnmb silencing protects against hyperoxia-induced acute lung injury by inhibition of mitochondrial-mediated apoptosis.

Background: Hyperoxia-induced acute lung injury (HALI) is a complication to ventilation in patients with respiratory failure, which can lead to acute inflammatory lung injury and chronic lung disease. The aim of this study was to integrate bioinformatics analysis to identify key genes associated with HALI and validate their role in H2O2-induced cell injury model.Methods: Integrated bioinformatics analysis was performed to screen vital genes involved in hyperoxia-induced lung injury (HLI). CCK-8 and flow cytometry assays were performed to assess cell viability and apoptosis. Western blotting was performed to assess protein expression.Results: In this study, glycoprotein non-metastatic melanoma protein B (Gpnmb) was identified as a key gene in HLI by integrated bioinformatics analysis of 4 Gene Expression Omnibus (GEO) datasets (GSE97804, GSE51039, GSE76301 and GSE87350). Knockdown of Gpnmb increased cell viability and decreased apoptosis in H2O2-treated MLE-12 cells, suggesting that Gpnmb was a proapoptotic gene during HALI. Western blotting results showed that knockdown of Gpnmb reduced the expression of Bcl-2 associated X (BAX) and cleaved-caspase 3, and increased the expression of Bcl-2 in H2O2 treated MLE-12 cells. Furthermore, Gpnmb knockdown could significantly reduce reactive oxygen species (ROS) generation and improve the mitochondrial membrane potential.Conclusion: The present study showed that knockdown of Gpnmb may protect against HLI by repressing mitochondrial-mediated apoptosis.

BCL-XL regulates the timing of mitotic apoptosis independently of BCL2 and MCL1 compensation.

Mitotic catastrophe induced by prolonged mitotic arrest is a major anticancer strategy. Although antiapoptotic BCL2-like proteins, including BCL-XL, are known to regulate apoptosis during mitotic arrest, adaptive changes in their expression can complicate loss-of-function studies. Our studies revealed compensatory alterations in the expression of BCL2 and MCL1 when BCL-XL is either downregulated or overexpressed. To circumvent their reciprocal regulation, we utilized a degron-mediated system to acutely silence BCL-XL just before mitosis. Our results show that in epithelial cell lines including HeLa and RPE1, BCL-XL and BCL2 acted collaboratively to suppress apoptosis during both unperturbed cell cycle and mitotic arrest. By tagging BCL-XL and BCL2 with a common epitope, we estimated that BCL-XL was less abundant than BCL2 in the cell. Nonetheless, BCL-XL played a more prominent antiapoptotic function than BCL2 during interphase and mitotic arrest. Loss of BCL-XL led to mitotic cell death primarily through a BAX-dependent process. Furthermore, silencing of BCL-XL led to the stabilization of MCL1, which played a significant role in buffering apoptosis during mitotic arrest. Nevertheless, even in a MCL1-deficient background, depletion of BCL-XL accelerated mitotic apoptosis. These findings underscore the pivotal involvement of BCL-XL in controlling timely apoptosis during mitotic arrest, despite adaptive changes in the expression of other BCL2-like proteins.

Bcl-xL targeting eliminates ageing tumor-promoting neutrophils and inhibits lung tumor growth.

Elevated peripheral blood and tumor-infiltrating neutrophils are often associated with a poor patient prognosis. However, therapeutic strategies to target these cells are difficult to implement due to the life-threatening risk of neutropenia. In a genetically engineered mouse model of lung adenocarcinoma, tumor-associated neutrophils (TAN) demonstrate tumor-supportive capacities and have a prolonged lifespan compared to circulating neutrophils. Here, we show that tumor cell-derived GM-CSF triggers the expression of the anti-apoptotic Bcl-xL protein and enhances neutrophil survival through JAK/STAT signaling. Targeting Bcl-xL activity with a specific BH3 mimetic, A-1331852, blocked the induced neutrophil survival without impacting their normal lifespan. Specifically, oral administration with A-1331852 decreased TAN survival and abundance, and reduced tumor growth without causing neutropenia. We also show that G-CSF, a drug used to combat neutropenia in patients receiving chemotherapy, increased the proportion of young TANs and augmented the anti-tumor effect resulting from Bcl-xL blockade. Finally, our human tumor data indicate the same role for Bcl-xL on pro-tumoral neutrophil survival. These results altogether provide preclinical evidence for safe neutrophil targeting based on their aberrant intra-tumor longevity.

ATP1A1/BCL2L1 predicts the response of myelomonocytic and monocytic acute myeloid leukemia to cardiac glycosides.

Myelomonocytic and monocytic acute myeloid leukemia (AML) subtypes are intrinsically resistant to venetoclax-based regimens. Identifying targetable vulnerabilities would limit resistance and relapse. We previously documented the synergism of venetoclax and cardiac glycoside (CG) combination in AML. Despite preclinical evidence, the repurposing of cardiac glycosides (CGs) in cancer therapy remained unsuccessful due to a lack of predictive biomarkers. We report that the ex vivo response of AML patient blasts and the in vitro sensitivity of established cell lines to the hemi-synthetic CG UNBS1450 correlates with the ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1)/BCL2 like 1 (BCL2L1) expression ratio. Publicly available AML datasets identify myelomonocytic/monocytic differentiation as the most robust prognostic feature, along with core-binding factor subunit beta (CBFB), lysine methyltransferase 2A (KMT2A) rearrangements, and missense Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Mechanistically, BCL2L1 protects from cell death commitment induced by the CG-mediated stepwise triggering of ionic perturbation, protein synthesis inhibition, and MCL1 downregulation. In vivo, CGs showed an overall tolerable profile while impacting tumor growth with an effect ranging from tumor growth inhibition to regression. These findings suggest a predictive marker for CG repurposing in specific AML subtypes.

First-in-Human Study with Preclinical Data of BCL-2/BCL-xL Inhibitor Pelcitoclax in Locally Advanced or Metastatic Solid Tumors.

PURPOSE: B-cell lymphoma-extra-large (BCL-xL) regulates apoptosis and is an attractive anticancer therapeutic target. However, BCL-xL inhibition also kills mature platelets, hampering clinical development. Using an innovative prodrug strategy, we have developed pelcitoclax (APG-1252), a potent, dual BCL-2 and BCL-xL inhibitor. Aims of this study were to characterize the antitumor activity and safety of pelcitoclax and explore its underlying mechanisms of action (MOA). PATIENTS AND METHODS: Cell line-derived xenograft and patient-derived xenograft (PDX) models were tested to evaluate antitumor activity and elucidate MOA. Subjects (N = 50) with metastatic small-cell lung cancer and other solid tumors received intravenous pelcitoclax once or twice weekly. Primary outcome measures were safety and tolerability; preliminary efficacy (responses every 2 cycles per RECIST version 1.1) represented a secondary endpoint. RESULTS: Pelcitoclax exhibited strong BAX/BAK‒dependent and caspase-mediated antiproliferative and apoptogenic activity in various cancer cell lines. Consistent with cell-based apoptogenic activity, pelcitoclax disrupted BCL-xL:BIM and BCL-xL:PUMA complexes in lung and gastric cancer PDX models. Levels of BCL-xL complexes correlated with tumor growth inhibition by pelcitoclax. Combined with taxanes, pelcitoclax enhanced antitumor activity by downregulating antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Importantly, pelcitoclax was well tolerated and demonstrated preliminary therapeutic efficacy, with overall response and disease control rates of 6.5% and 30.4%, respectively. Most common treatment-related adverse events included transaminase elevations and reduced platelets that were less frequent with a once-weekly schedule. CONCLUSIONS: Our data demonstrate that pelcitoclax has antitumor activity and is well tolerated, supporting its further clinical development for human solid tumors, particularly combined with agents that downregulate MCL-1.

BH3 mimetics and azacitidine show synergistic effects on juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is an aggressive hematopoietic disorder of infancy and early childhood driven by constitutively active RAS signaling and characterized by abnormal proliferation of the granulocytic-monocytic blood cell lineage. Most JMML patients require hematopoietic stem cell transplantation for cure, but the risk of relapse is high for some JMML subtypes. Azacitidine was shown to effectively reduce leukemic burden in a subset of JMML patients. However, variable response rates to azacitidine and the risk of drug resistance highlight the need for novel therapeutic approaches. Since RAS signaling is known to interfere with the intrinsic apoptosis pathway, we combined various BH3 mimetic drugs with azacitidine in our previously established patient-derived xenograft model. We demonstrate that JMML cells require both MCL-1 and BCL-XL for survival, and that these proteins can be effectively targeted by azacitidine and BH3 mimetic combination treatment. In vivo azacitidine acts via downregulation of antiapoptotic MCL-1 and upregulation of proapoptotic BH3-only. The combination of azacitidine with BCL-XL inhibition was superior to BCL-2 inhibition in eliminating JMML cells. Our findings emphasize the need to develop clinically applicable MCL-1 or BCL-XL inhibitors in order to enable novel combination therapies in JMML refractory to standard therapy.

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.

Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis.

Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis.

Chemical modulation of cytosolic BAX homodimer potentiates BAX activation and apoptosis.

The BCL-2 family protein BAX is a major regulator of physiological and pathological cell death. BAX predominantly resides in the cytosol in a quiescent state and upon stress, it undergoes conformational activation and mitochondrial translocation leading to mitochondrial outer membrane permeabilization, a critical event in apoptosis execution. Previous studies reported two inactive conformations of cytosolic BAX, a monomer and a dimer, however, it remains unclear how they regulate BAX. Here we show that, surprisingly, cancer cell lines express cytosolic inactive BAX dimers and/or monomers. Expression of inactive dimers, results in reduced BAX activation, translocation and apoptosis upon pro-apoptotic drug treatments. Using the inactive BAX dimer structure and a pharmacophore-based drug screen, we identify a small-molecule modulator, BDM19 that binds and activates cytosolic BAX dimers and prompts cells to apoptosis either alone or in combination with BCL-2/BCL-XL inhibitor Navitoclax. Our findings underscore the role of the cytosolic inactive BAX dimer in resistance to apoptosis and demonstrate a strategy to potentiate BAX-mediated apoptosis.