Contribution of A1 to macrophage survival in cooperation with MCL-1 and BCL-XL in a murine cell model of myeloid differentiation.

Myeloid cells are the first line of defence against pathogens. Mitochondrial apoptosis signalling is a crucial regulator of myeloid cell lifespan and modulates the function of myeloid cells. The anti-apoptotic protein BCL-2-family protein BCL2A1/A1/BFL-1 is strongly upregulated in inflammation in macrophages. We analysed the contribution of A1 to apoptosis regulation in a conditional system of in vitro differentiation of murine macrophages from immortalised progenitors. We disabled the expression of A1 by targeting all murine A1 isoforms in the genome. Specific inhibitors were used to inactivate other anti-apoptotic proteins. Macrophage progenitor survival mainly depended on the anti-apoptotic proteins MCL-1, BCL-XL and A1 but not BCL-2. Deletion of A1 on its own had little effect on progenitor cell survival but was sensitised to cell death induction when BCL-XL or MCL-1 was neutralised. In progenitors, A1 was required for survival in the presence of the inflammatory stimulus LPS. Differentiated macrophages were resistant to inhibition of single anti-apoptotic proteins, but A1 was required to protect macrophages against inhibition of either BCL-XL or MCL-1; BCL-2 only had a minor role in these cells. Cell death by neutralisation of anti-apoptotic proteins completely depended on BAX with a small contribution of BAK only in progenitors in the presence of LPS. A1 and NOXA appeared to stabilise each other at the posttranscriptional level suggesting direct binding. Co-immunoprecipitation experiments showed the binding of A1 to NOXA and BIM. Interaction between A1 and Noxa may indirectly prevent neutralisation and destabilization of MCL-1. Our findings suggest a unique role for A1 as a modulator of survival in the macrophage lineage in concert with MCL-1 and BCL-XL, especially in a pro-inflammatory environment.

Induction of Apoptotic Signaling Pathways by 3' methyl ATP in Different Malignant Cells: in vitro Study.

Extracellular ATP is a dynamic signaling molecule that modulates myriad of cellular functions through P2 purinergic receptors activation and is cytotoxic to a variety of cells at high concentration. But the mechanism of this extracellular ATP/ATP analogs- elicited cytotoxicity is not fully understood. In this study we aim to investigate whether there is differential sensitivity towards induction of apoptosis by ATP analogs (2'-Me ATP and 3'-Me ATP) and its effect on receptor mediated or extrinsic and mitochondria mediated or intrinsic apoptotic signaling pathways. Our findings demonstrated that the IC50 values for 2'-Me ATP and 3'-Me ATP were 3mM and 2mM, respectively, in Hep2, and SiHa cells. The downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, along with a significant increase in the expression of the pro-apoptotic protein Bax (p<0.05), indicated the involvement of both pro- and anti-apoptotic factors in HeP2 cells, whereas in SiHa cells, a downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL was observed, whereas the expression level of the pro-apoptotic protein Bax remained unaffected. Furthermore, an upregulation of p53 and apoptosis-inducing factor (AIF) was observed in HeP2 cells (p<0.05) whereas, an upregulation of p53 was observed while no change was seen on the level of apoptosis inducing factor (AIF) was observed in SiHa cells. Additionally, there was a notable rise in caspase-3 and -9 activities, PARP cleavage, and the release of cytochrome c (p<0.05) from the mitochondria to the cytosol in both cells. Collectively, our study suggests that 3'-Me ATP induces apoptosis in Hep2 and SiHa cells through the intrinsic mitochondrial pathway.

Proteasome inhibition induces apoptosis through simultaneous inactivation of MCL-1/BCL-XL by NOXA independent of CHOP and JNK pathways.

Proteasome inhibitors have been employed in the treatment of relapsed multiple myeloma and mantle cell lymphoma. The observed toxicity caused by proteasome inhibitors is a universal phenotype in numerous cancer cells with different sensitivity. In this study, we investigate the conserved mechanisms underlying the toxicity of the proteasome inhibitor bortezomib using gene editing approaches. Our findings utilizing different caspase knocking out cells reveal that bortezomib induces classic intrinsic apoptosis by activating caspase-9 and caspase-3/7, leading to pore-forming protein GSDME cleavage and subsequent lytic cell death or called secondary necrosis, a phenotype also observed in many apoptosis triggers like TNFα plus CHX, DTT and tunicamycin treatment in HeLa cells. Furthermore, through knocking out of nearly all BH3-only proteins including BIM, BAD, BID, BMF and PUMA, we demonstrate that NOXA is the sole BH3-only protein responsible for bortezomib-induced apoptosis. Of note, NOXA is well known for selectively binding to MCL-1 and A1, but our studies utilizing different BH3 mimetics as well as immunoprecipitation assays indicate that, except for the constitutive interaction of NOXA with MCL-1, the accumulation of NOXA after bortezomib treatment allows it to interact with BCL-XL, then simultaneous relieving suppression on apoptosis by both anti-apoptotic proteins BCL-XL and MCL-1. In addition, though bortezomib-induced significant ER stress and JNK activation were observed in the study, further genetic depletion experiments prove that bortezomib-induced apoptosis occurs independently of ER stress-related apoptosis factor CHOP and JNK. In summary, these results provide a solid conclusion about the critical role of NOXA in inactivation of BCL-XL except MCL-1 in bortezomib-induced apoptosis.

Integrative single-cell RNA-seq and spatial transcriptomics analyses reveal diverse apoptosis-related gene expression profiles in EGFR-mutated lung cancer.

In EGFR-mutated lung cancer, the duration of response to tyrosine kinase inhibitors (TKIs) is limited by the development of acquired drug resistance. Despite the crucial role played by apoptosis-related genes in tumor cell survival, how their expression changes as resistance to EGFR-TKIs emerges remains unclear. Here, we conduct a comprehensive analysis of apoptosis-related genes, including BCL-2 and IAP family members, using single-cell RNA sequence (scRNA-seq) and spatial transcriptomics (ST). scRNA-seq of EGFR-mutated lung cancer cell lines captures changes in apoptosis-related gene expression following EGFR-TKI treatment, most notably BCL2L1 upregulation. scRNA-seq of EGFR-mutated lung cancer patient samples also reveals high BCL2L1 expression, specifically in tumor cells, while MCL1 expression is lower in tumors compared to non-tumor cells. ST analysis of specimens from transgenic mice with EGFR-driven lung cancer indicates spatial heterogeneity of tumors and corroborates scRNA-seq findings. Genetic ablation and pharmacological inhibition of BCL2L1/BCL-XL overcome or delay EGFR-TKI resistance. Overall, our findings indicate that BCL2L1/BCL-XL expression is important for tumor cell survival as EGFR-TKI resistance emerges.

APG-1252 combined with Cabozantinib inhibits hepatocellular carcinoma by suppressing MEK/ERK and CREB/Bcl-xl pathways.

BACKGROUND AND PURPOSE: Liver cancer is the fourth leading cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the most common type of primary liver cancer. APG-1252 is a small molecule inhibitor targeting Bcl-2 and Bcl-xl. However, its anti-tumor effects in HCC, alone or in combination with Cabozantinib, have not been extensively studied. EXPERIMENTAL: Approach: TCGA database analysis was used to analysis the gene expression levels of Bcl-2 and Bcl-xl in HCC tissues. Western blot was employed to detect the protein expression levels. And the inhibitory effects of APG-1252 and Cabozantinib on the proliferation of HCC cell lines was detected by CCK-8. The effect on the migration and invasion of HCC cells was verified by transwell assay. Huh7 xenograft model in nude mice was used to investigate the combination antitumor effect in vivo. KEY RESULTS: Our study demonstrated that APG-1252 monotherapy inhibited the proliferation and migration ability of HCC cells, and induced HCC cells apoptosis. The combination of APG-1252 and Cabozantinib showed significant synergistic antitumor effects. Furthermore, the in vivo experiment demonstrated that the combination therapy exerted a synergistic effect in delaying tumor growth, notably downregulating MEK/ERK phosphorylation levels. In terms of mechanism, Cabozantinib treatment caused an increase in the phosphorylation levels of CREB and Bcl-xl proteins, while the combination with APG-1252 mitigated this effect, thereby enhanced the antitumor effect of Cabozantinib. CONCLUSION AND IMPLICATIONS: Our findings suggest that APG-1252 in combination with Cabozantinib offers a more effective treatment strategy for HCC patients, warranting further clinical investigation.

Comparing the BAD Protein Interactomes in 2D and 3D Cell Culture Using Proximity Labeling.

Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.

Small Cell Lung Carcinoma Cells Depend on KIF11 for Survival.

Few efficacious treatment options are available for patients with small cell lung carcinoma (SCLC), indicating the need to develop novel therapeutic approaches. In this study, we explored kinesin family member 11 (KIF11), a potential therapeutic target in SCLC. An analysis of publicly available data suggested that KIF11 mRNA expression levels are significantly higher in SCLC tissues than in normal lung tissues. When KIF11 was targeted by RNA interference or a small-molecule inhibitor (SB743921) in two SCLC cell lines, Lu-135 and NCI-H69, cell cycle progression was arrested at the G2/M phase with complete growth suppression. Further work suggested that the two cell lines were more significantly affected when both KIF11 and BCL2L1, an anti-apoptotic BCL2 family member, were inhibited. This dual inhibition resulted in markedly decreased cell viability. These findings collectively indicate that SCLC cells are critically dependent on KIF11 activity for survival and/or proliferation, as well as that KIF11 inhibition could be a new strategy for SCLC treatment.

Preferred inhibition of pro-apoptotic Bak by BclxL via a two-step mechanism.

Bak is a pore-forming Bcl2 protein that induces apoptosis at the outer mitochondrial membrane, which can either proceed via Bak oligomerization or be inhibited by anti-apoptotic Bcl2 proteins, such as BclxL. BclxL is very efficient in inhibiting Bak pore formation, but the mechanistic basis of this preferred interaction has remained enigmatic. Here, we identify Bakα1 as a second binding site for BclxL and show that it specifically interacts with the Bcl2-homology (BH)3 binding groove of BclxL. The affinity between BclxL and Bakα1 is weaker than with Bak-BH3, suggesting that Bakα1, being exposed early in the pore-forming trajectory, transiently captures BclxL, which subsequently transitions to the proximal BH3 site. Bak variants where the initial transient interaction with BclxL is modulated show a markedly altered response to BclxL inhibition. This work contributes to a better mechanistic understanding of the fine-tuned interactions between different players of the Bcl2 protein family.

Altered RNA export by SF3B1 mutants confers sensitivity to nuclear export inhibition.

SF3B1 mutations frequently occur in cancer yet lack targeted therapies. Clinical trials of XPO1 inhibitors, selinexor and eltanexor, in high-risk myelodysplastic neoplasms (MDS) revealed responders were enriched with SF3B1 mutations. Given that XPO1 (Exportin-1) is a nuclear exporter responsible for the export of proteins and multiple RNA species, this led to the hypothesis that SF3B1-mutant cells are sensitive to XPO1 inhibition, potentially due to altered splicing. Subsequent RNA sequencing after XPO1 inhibition in SF3B1 wildtype and mutant cells showed increased nuclear retention of RNA transcripts and increased alternative splicing in the SF3B1 mutant cells particularly of genes that impact apoptotic pathways. To identify novel drug combinations that synergize with XPO1 inhibition, a forward genetic screen was performed with eltanexor treatment implicating anti-apoptotic targets BCL2 and BCLXL, which were validated by functional testing in vitro and in vivo. These targets were tested in vivo using Sf3b1K700E conditional knock-in mice, which showed that the combination of eltanexor and venetoclax (BCL2 inhibitor) had a preferential sensitivity for SF3B1 mutant cells without excessive toxicity. In this study, we unveil the mechanisms underlying sensitization to XPO1 inhibition in SF3B1-mutant MDS and preclinically rationalize the combination of eltanexor and venetoclax for high-risk MDS.

Synergy of retinoic acid and BH3 mimetics in MYC(N)-driven embryonal nervous system tumours.

BACKGROUND: Certain paediatric nervous system malignancies have dismal prognoses. Retinoic acid (RA) is used in neuroblastoma treatment, and preclinical data indicate potential benefit in selected paediatric brain tumour entities. However, limited single-agent efficacy necessitates combination treatment approaches. METHODS: We performed drug sensitivity profiling of 76 clinically relevant drugs in combination with RA in 16 models (including patient-derived tumouroids) of the most common paediatric nervous system tumours. Drug responses were assessed by viability assays, high-content imaging, and apoptosis assays and RA relevant pathways by RNAseq from treated models and patient samples obtained through the precision oncology programme INFORM (n = 2288). Immunoprecipitation detected BCL-2 family interactions, and zebrafish embryo xenografts were used for in vivo efficacy testing. RESULTS: Group 3 medulloblastoma (MBG3) and neuroblastoma models were highly sensitive to RA treatment. RA induced differentiation and regulated apoptotic genes. RNAseq analysis revealed high expression of BCL2L1 in MBG3 and BCL2 in neuroblastomas. Co-treatments with RA and BCL-2/XL inhibitor navitoclax synergistically decreased viability at clinically achievable concentrations. The combination of RA with navitoclax disrupted the binding of BIM to BCL-XL in MBG3 and to BCL-2 in neuroblastoma, inducing apoptosis in vitro and in vivo. CONCLUSIONS: RA treatment primes MBG3 and NB cells for apoptosis, triggered by navitoclax cotreatment.

Comparative analysis of Bcl-2 family protein overexpression in CAR T cells alone and in combination with BH3 mimetics.

Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.

Low BCL-xL expression in triple-negative breast cancer cells favors chemotherapy efficacy, and this effect is limited by cancer-associated fibroblasts.

Triple negative breast cancers (TNBC) present a poor prognosis primarily due to their resistance to chemotherapy. This resistance is known to be associated with elevated expression of certain anti-apoptotic members within the proteins of the BCL-2 family (namely BCL-xL, MCL-1 and BCL-2). These regulate cell death by inhibiting pro-apoptotic protein activation through binding and sequestration and they can be selectively antagonized by BH3 mimetics. Yet the individual influences of BCL-xL, MCL-1, and BCL-2 on the sensitivity of TNBC cells to chemotherapy, and their regulation by cancer-associated fibroblasts (CAFs), major components of the tumor stroma and key contributors to therapy resistance remain to be delineated. Using gene editing or BH3 mimetics to inhibit anti-apoptotic BCL-2 family proteins in TNBC line MDA-MB-231, we show that BCL-xL and MCL-1 promote cancer cell survival through compensatory mechanisms. This cell line shows limited sensitivity to chemotherapy, in line with the clinical resistance observed in TNBC patients. We elucidate that BCL-xL plays a pivotal role in therapy response, as its depletion or pharmacological inhibition heightened chemotherapy effectiveness. Moreover, BCL-xL expression is associated with chemotherapy resistance in patient-derived tumoroids where its pharmacological inhibition enhances ex vivo response to chemotherapy. In a co-culture model of cancer cells and CAFs, we observe that even in a context where BCL-xL reduced expression renders cancer cells more susceptible to chemotherapy, those in contact with CAFs display reduced sensitivity to chemotherapy. Thus CAFs exert a profound pro-survival effect in breast cancer cells, even in a setting highly favoring cell death through combined chemotherapy and absence of the main actor of chemoresistance, BCL-xL.

p85α deficiency alleviates ischemia-reperfusion injury by promoting cardiomyocyte survival.

Myocardial ischemia-reperfusion (I/R) injury is a prevalent cause of myocardial injury, involving a series of interconnected pathophysiological processes. However, there is currently no clinical therapy for effectively mitigating myocardial I/R injury. Here, we show that p85α protein levels increase in response to I/R injury through a comprehensive analysis of cardiac proteomics, and confirm this in the I/R-injured murine heart and failing human myocardium. Genetic inhibition of p85α in mice activates the Akt-GSK3ß/Bcl-x(L) signaling pathway and ameliorates I/R-induced cardiac dysfunction, apoptosis, inflammation, and mitochondrial dysfunction. p85α silencing in cardiomyocytes alleviates hypoxia-reoxygenation (H/R) injury through activating the Akt-GSK3ß/Bcl-x(L) signaling pathway, while its overexpression exacerbates the damage. Mechanistically, the interaction between MG53 and p85α triggers the ubiquitination and degradation of p85α, consequently enhancing Akt phosphorylation and ultimately having cardioprotective effects. Collectively, our findings reveal that substantial reduction of p85α and subsequently activated Akt signaling have a protective effect against cardiac I/R injury, representing an important therapeutic strategy for mitigating myocardial damage.

BCL2L1 is regulated by the lncRNA MIR4435-2HG-miR-513a-5p-BCL2L1 ceRNA axis and serves as a biomarker for pancreatic adenocarcinoma treatment and prognosis.

Pancreatic adenocarcinoma (PAAD) is one of the most malignant cancers. After escaping death, cancer cells are made more metastatic, aggressive, and also drug-resistant through anoikis resistance. The aim of this study is to explore the molecular mechanisms of anoikis-related genes in PAAD and to identify potential key biomarkers. We integrated information about PAAD from The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) databases and identified anoikis-related gene BCL2L1 by survival analysis, univariate Cox regression analysis, and multifactorial Cox regression analysis. Various bioinformatics approaches showed that BCL2L1 was a valuable prognostic marker that might be involved in PAAD development and progression through different mechanisms, including cancer intervention, genomic heterogeneity, and RNA modifications. Our analysis showed that BCL2L1 expression also closely correlates with the expression of various immune checkpoint inhibitors. In particular, we found that long non-coding RNA MIR4435-2HG acted as ceRNA sponging miR-513a-5p to promote the expression of BCL2L1, thereby promoting pancreatic cancer cells proliferation. In conclusion, BCL2L1 expression regulated by the MIR4435-2HG-miR-513a-5p-BCL2L1 ceRNA axis might be used as a biomarker for cancer prognosis, treatment selection, and follow-up in PAAD patients.

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.

Blockage of BCL-XL overcomes venetoclax resistance across BCL2+ lymphoid malignancies irrespective of BIM status.

ABSTRACT: Venetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which call for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient-derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL2L11/BIM status.

Involvement of Janus kinase-dependent Bcl-xL overexpression in steroid resistance of group 2 innate lymphoid cells in asthma.

The mechanisms underlying the development of steroid resistance in asthma remain unclear. To establish whether as well as the mechanisms by which the activation of Janus kinases (JAKs) is involved in the development of steroid resistance in asthma, murine steroid-resistant models of the proliferation of group 2 innate lymphoid cells (ILC2s) in vitro and asthmatic airway inflammation in vivo were analysed. ILC2s in the lungs of BALB/c mice were sorted and then incubated with IL-33, thymic stromal lymphopoietin (TSLP), and/or IL-7 with or without dexamethasone (10 nM), the pan-JAK inhibitor, delgocitinib (1-10 000 nM), and/or the Bcl-xL inhibitor, navitoclax (1-100 nM), followed by the detection of viable and apoptotic cells. The anti-apoptotic factor, Bcl-xL was detected in ILC2s by flow cytometry. As a steroid-resistant asthma model, ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at a high dose of 500 µg four times. Dexamethasone (1 mg/kg, i.p.), delgocitinib (3-30 mg/kg, p.o.), or navitoclax (30 mg/kg, p.o.) was administered during the challenges. Cellular infiltration into the lungs was analysed by flow cytometry. Airway remodelling was histologically evaluated. The following results were obtained. (1) Cell proliferation concomitant with a decrease in apoptotic cells was induced when ILC2s were cultured with TSLP and/or IL-7, and was potently inhibited by dexamethasone. In contrast, when the culture with TSLP and IL-7 was performed in the presence of IL-33, the proliferative response exhibited steroid resistance. Steroid-resistant ILC2 proliferation was suppressed by delgocitinib in a concentration-dependent manner. (2) The culture with IL-33, TSLP, and IL-7 induced the overexpression of Bcl-xL, which was clearly inhibited by delgocitinib, but not by dexamethasone. When ILC2s were treated with navitoclax, insensitivity to dexamethasone was significantly cancelled. (3) The development of airway remodelling and the infiltration of ILC2s into the lungs in the asthma model were not suppressed by dexamethasone, but were dose-dependently inhibited by delgocitinib. Combination treatment with dexamethasone and either delgocitinib or navitoclax synergistically suppressed these responses. Therefore, JAKs appear to play significant roles in the induction of steroid resistance by up-regulating Bcl-xL in ILC2s. The inhibition of JAKs and Bcl-xL has potential as pharmacotherapy for steroid-resistant asthma, particularly that mediated by ILC2s.

Profiling of BCLxL Protein Complexes in Non-Small Cell Lung Cancer Cells via Multiplexed Single-Molecule Pull-Down and Co-Immunoprecipitation.

We introduce multiplexed single-molecule pull-down and co-immunoprecipitation, named m-SMPC, an analysis tool for profiling multiple protein complexes within a single reaction chamber using single-molecule fluorescence imaging. We employed site-selective conjugation of biotin and fluorescent dye directly onto the monoclonal antibodies, which completed an independent sandwich immunoassay without the issue of host cross-reactivity. We applied this technique to profile endogenous B-cell lymphoma extra-large (BCLxL) complexes in non-small cell lung cancer (NSCLC) cells. Up to three distinct BCLxL complexes were successfully detected simultaneously within a single reaction chamber without fluorescence signal crosstalk. Notably, the NSCLC cell line EBC-1 exhibited high BCLxL-BAX and BCLxL-BAK levels, which closely paralleled a strong response to the BCLxL inhibitor A-1331852. This streamlined method offers the potential for quantitative biomarkers derived from protein complex profiling, paving the way for their application in protein complex-targeted therapies.

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.

Combination p53 activation and BCL-xL/BCL-2 inhibition as a therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia.

Due to the rarity of TP53 mutations in acute lymphoblastic leukemia (ALL), p53 re-activation by antagonism of the p53-MDM2 interaction represents a potential therapeutic strategy for the majority of ALL. Here, we demonstrate the potent antileukemic activity of the MDM2 antagonist idasanutlin in high-risk and relapsed ex vivo coculture models of TP53 wildtype ALL (n = 40). Insufficient clinical responses to monotherapy MDM2 inhibitors in other cancers prompted us to explore optimal drugs for combination therapy. Utilizing high-throughput combination screening of 1971 FDA-approved and clinically advanced compounds, we identified BCL-xL/BCL-2 inhibitor navitoclax as the most promising idasanutlin combination partner. The idasanutlin-navitoclax combination was synergistically lethal to prognostically-poor, primary-derived and primary patient blasts in ex vivo coculture, and reduced leukemia burden in two very high-risk ALL xenograft models at drug concentrations safely attained in patients; in fact, the navitoclax plasma concentrations were equivalent to those attained in contemporary "low-dose" navitoclax clinical trials. We demonstrate a preferential engagement of cell death over G1 cell cycle arrest, mechanistically implicating MCL-1-binding pro-apoptotic sensitizer NOXA. The proposed combination of two clinical-stage compounds independently under clinical evaluation for ALL is of high clinical relevance and warrants consideration for the treatment of patients with high-risk and relapsed ALL.