Bcl-2 dependent modulation of Hippo pathway in cancer cells.

INTRODUCTION: Bcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2. METHODS: We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts. RESULTS: We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation. CONCLUSIONS: We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

[Curcumin Combined with Thalidomide Inhibits Proliferation of KG-1 Cells and Its Related Mechanisms].

OBJECTIVE: To investigate the effects of curcumin combined with thalidomide on the proliferation and apoptosis of acute myeloid leukemia KG-1 cells, and its correlation with B-cell lymphoma-xL (Bcl-xL), signal transducer and activator of transcription 3 (STAT3). METHODS: MTT assay was used to detect the proliferation of KG-1 cells and screen the optimal combined concentration of curcumin and thalidomide. The effects of curcumin, thalidomide and their combination on the proliferation and apoptosis of KG-1 cells were analyzed by MTT method and flow cytometry, respectively. The mRNA expression levels of STAT3 and Bcl-xL in single-drug group, two-drug combination group and control group (untreated cells) were detected by real-time quantitative PCR. RESULTS: Both curcumin and thalidomide inhibited the proliferation of KG-1 cells in a concentration-dependent manner in the range of 20-100 µmol/L (r =0.657, r =0.681). The IC50 value of curcumin and thalidomide at 48 h was (42.07±0.50) µmol/L and (57.01±2.39) µmol/L, respectively. The cell proliferation inhibition rate of curcumin (40 µmol/L) + thalidomide (60 µmol/L) was (86.67±1.53)%, which was significantly higher than (51.67±1.15)% of curcumin (40 µmol/L) and (55.33±1.53)% of thalidomide (60 µmol/L) (both P < 0.05). Treated with curcumin and thalidomide alone or in combination, the apoptosis rate of KT-1 cells was (18.67±2.08)%, (21.33±2.52)%, and (46.67±1.53)%, respectively, which was significantly higher than (0.72±0.03)% of control group (all P < 0.05). The cell apoptosis rate of two-drug combination group was significantly higher than that of single-drug group (both P < 0.05). Compared with the control group, the mRNA expressions of STAT3 and Bcl-xL in single-drug group, two-drug combination group were significantly decreased (both P < 0.05). Compared with single-drug group, the mRNA expressions of STAT3 and Bcl-xL in two-drug combination group were also significantly decreased (both P < 0.05). CONCLUSION: Curcumin combined with thalidomide can synergistically down-regulate the expression of STAT3 and Bcl-xL, inhibit the proliferation of KG-1 cells, and induce apoptosis.

Peritoneal B1 and B2 cells respond differently to LPS and IL-21 stimulation.

Peritoneal B cells can be divided into B1 cells (CD11b+CD19+) and B2 cells (CD11b-CD19+) based on CD11b expression. B1 cells play a crucial role in the innate immune response by producing natural antibodies and cytokines. B2 cells share similar traits with B1 cells, influenced by the peritoneal environment. However, the response of both B1 and B2 cells to the same stimuli in the peritoneum remains uncertain. We isolated peritoneal B1 and B2 cells from mice and assessed differences in Interleukin-10(IL-10) secretion, apoptosis, and surface molecule expression following exposure to LPS and Interleukin-21(IL-21). Our findings indicate that B1 cells are potent IL-10 producers, possessing surface molecules with an IgMhiCD43+CD21low profile, and exhibit a propensity for apoptosis in vitro. Conversely, B2 cells exhibit lower IL-10 production and surface markers characterized as IgMlowCD43-CD21hi, indicative of some resistance to apoptosis. LPS stimulates MAPK phosphorylation in B1 and B2 cells, causing IL-10 production. Furthermore, LPS inhibits peritoneal B2 cell apoptosis by enhancing Bcl-xL expression. Conversely, IL-21 has no impact on IL-10 production in these cells. Nevertheless, impeding STAT3 phosphorylation permits IL-21 to increase IL-10 production in peritoneal B cells. Moreover, IL-21 significantly raises apoptosis levels in these cells, a process independent of STAT3 phosphorylation and possibly linked to reduced Bcl-xL expression. This study elucidates the distinct functional and response profiles of B1 and B2 cells in the peritoneum to stimuli like LPS and IL-21, highlighting their differential roles in immunological responses and B cell diversity.

p53-Armed Oncolytic Virotherapy Improves Radiosensitivity in Soft-Tissue Sarcoma by Suppressing BCL-xL Expression.

Soft-tissue sarcoma (STS) is a heterogeneous group of rare tumors originating predominantly from the embryonic mesoderm. Despite the development of combined modalities including radiotherapy, STSs are often refractory to antitumor modalities, and novel strategies that improve the prognosis of STS patients are needed. We previously demonstrated the therapeutic potential of two telomerase-specific replication-competent oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702, in human STS cells. Here, we demonstrate in vitro and in vivo antitumor effects of OBP-702 in combination with ionizing radiation against human STS cells (HT1080, NMS-2, SYO-1). OBP-702 synergistically promoted the antitumor effect of ionizing radiation in the STS cells by suppressing the expression of B-cell lymphoma-X large (BCL-xL) and enhancing ionizing radiation-induced apoptosis. The in vivo experiments demonstrated that this combination therapy significantly suppressed STS tumors' growth. Our results suggest that OBP-702 is a promising antitumor reagent for promoting the radiosensitivity of STS tumors.

Fucoxanthin induces human melanoma cytotoxicity by thwarting the JAK2/STAT3/BCL-xL signaling axis.

Melanoma is the most lethal skin malignancy. Fucoxanthin is a marine carotenoid with significant anticancer activities. Intriguingly, Fucoxanthin's impact on human melanoma remains elusive. Signal Transducer and Activator of Transcription 3 (STAT3) represents a promising target in cancer therapy due to its persistent activation in various cancers, including melanoma. Herein, we revealed that Fucoxanthin is cytotoxic to human melanoma cell lines A2758 and A375 while showing limited cytotoxicity to normal human melanocytes. Apoptosis is a primary reason for Fucoxanthin's melanoma cytotoxicity, as the pan-caspase inhibitor z-VAD-fmk drastically abrogated Fucoxanthin-elicited clonogenicity blockage. Besides, Fucoxanthin downregulated tyrosine 705-phosphorylated STAT3 (p-STAT3 (Y705)), either inherently present in melanoma cells or inducible by interleukin 6 (IL-6) stimulation. Notably, ectopic expression of STAT3-C, a dominant-active STAT3 mutant, abolished Fucoxanthin-elicited melanoma cell apoptosis and clonogenicity inhibition, supporting the pivotal role of STAT3 blockage in Fucoxanthin's melanoma cytotoxicity. Moreover, Fucoxanthin lowered BCL-xL levels by blocking STAT3 activation, while ectopic BCL-xL expression rescued melanoma cells from Fucoxanthin-induced killing. Lastly, Fucoxanthin was found to diminish the levels of JAK2 with dual phosphorylation at tyrosine residues 1007 and 1008 in melanoma cells, suggesting that Fucoxanthin impairs STAT3 signaling by blocking JAK2 activation. Collectively, we present the first evidence that Fucoxanthin is cytotoxic selectively against human melanoma cells while sparing normal melanocytes. Mechanistically, Fucoxanthin targets the JAK2/STAT3/BCL-xL antiapoptotic axis to provoke melanoma cell death. This discovery implicates the potential application of Fucoxanthin as a chemopreventive or therapeutic strategy for melanoma management.

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.

AMPK inhibition sensitizes acute leukemia cells to BH3 mimetic-induced cell death.

BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.

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.

OGDH and Bcl-xL loss causes synthetic lethality in glioblastoma.

Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the α-ketoglutarate dehydrogenase (OGDH) gene, which encodes an enzyme that is part of the tricarboxylic acid (TCA) cycle, as essential for GBM growth. Moreover, by combining transcriptome and metabolite screening analyses, we discovered that loss of function of OGDH by the clinically validated drug compound CPI-613 was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3 mimetic, ABT263) in patient-derived xenografts as well neurosphere GBM cultures. CPI-613-mediated energy deprivation drove an integrated stress response with an upregulation of the BH3-only domain protein, Noxa, in an ATF4-dependent manner, as demonstrated by genetic loss-of-function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with disruption of the TCA cycle might be a treatment strategy for GBM.

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.

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.

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.

ZNF862 induces cytostasis and apoptosis via the p21-RB1 and Bcl-xL-Caspase 3 signaling pathways in human gingival fibroblasts.

OBJECTIVE: This study investigates the effects of ZNF862 on the proliferation and apoptosis of human gingival fibroblasts and their related mechanisms. BACKGROUND: As a major transcription factor family, zinc finger proteins (ZFPs) regulate cell differentiation, growth, and apoptosis through their conserved zinc finger motifs, which allow high flexibility and specificity in gene regulation. In our previous study, ZNF862 mutation was associated with hereditary gingival fibromatosis. Nevertheless, little is known about the biological function of ZNF862. Therefore, this study was aimed to reveal intracellular localization of ZNF862, the influence of ZNF862 on the growth and apoptosis of human gingival fibroblasts (HGFs) and its potential related mechanisms. METHODS: Immunohistochemistry, immunofluorescence staining, and western blotting were performed to determine the intracellular localization of ZNF862 in HGFs. HGFs were divided into three groups: ZNF862 overexpression group, ZNF862 interference group, and the empty vector control group. Then, the effects of ZNF862 on cell proliferation, migration, cell cycle, and apoptosis were evaluated. qRT-PCR and western blotting were performed to further explore the mechanism related to the proliferation and apoptosis of HGFs. RESULTS: ZNF862 was found to be localized in the cytoplasm of HGFs. In vitro experiments revealed that ZNF862 overexpression inhibited HGFs proliferation and migration, induced cell cycle arrest at the G0/G1-phase and apoptosis. Whereas, ZNF862 knockdown promoted HGFs proliferation and migration, accelerated the transition from the G0/G1 phase into the S and G2/M phase and inhibited cell apoptosis. Mechanistically, the effects of ZNF862 on HGFs proliferation and apoptosis were noted to be dependent on inhibiting the cyclin-dependent kinase inhibitor 1A (p21)-retinoblastoma 1 (RB1) signaling pathway and enhancing the B-cell lymphoma-extra-large (Bcl-xL)-Caspase 3 signaling pathway. CONCLUSION: Our results for the first time reveal that ZNF862 is localized in the cytoplasm of HGFs. ZNF862 can inhibit the proliferation of HGFs by inhibiting the p21-RB1 signaling pathway, and it also promotes the apoptosis of HGFs by enhancing the Bcl-xL-Caspase 3 signaling pathway.

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.

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.

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.

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.

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.