Induction Of Bcl-xL Research Articles

Differential expression pattern of Bcl-2 family members in B and T cells in systemic lupus erythematosus and rheumatoid arthritis

ObjectiveThis study aimed to evaluate the expression level of anti-apoptotic Bcl-2 family proteins in B and T cells in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in relation to disease activity and the effect of various Bcl-2 family inhibitors (BH3 mimetics) as potential treatment.MethodsWe included 14 SLE patients, 12 RA patients, and 13 healthy controls to study anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1 expression and cell survival in different B and T cell subsets using stimulation assays and intracellular flow cytometry. Effect of various BH3 mimetics was assessed by cell viability analyses.ResultsIn SLE, significant differences in Bcl-2 family members were confined to the B cell compartment with decreased induction of Bcl-XL (p ≤ 0.05) and Mcl-1 (p ≤ 0.001) upon CpG stimulation. In RA, we did not observe any differences in expression levels of Bcl-2 family proteins. Expression patterns did not correlate with disease activity apart from decreased induction of Mcl-1 in B cells in active SLE. After in vitro stimulation with CpG, plasmablasts were more viable after treatment with three different BH3 mimetics compared to naïve or memory B cells in control and patient cells. After activation, Mcl-1 inhibition was most effective in reducing plasmablast and T cell viability, however, less in patients than controls.ConclusionOur study provides evidence for the increased differential expression pattern of Bcl-2 family members in B and T cell subsets of patients with SLE compared to controls. Tested BH3 mimetics showed higher efficacy in controls compared to both autoimmune diseases, though nonsignificant due to low patient numbers.

Abstract 1864: An improved trispecific antibody (TsAb) platform for optimally engaging T cells for the treatment of solid tumor malignancies

Abstract T-cell recruiting bispecific antibodies have been shown to be clinically effective in treating hematological malignancies. However, their application against solid tumors is limited by several challenges, including a narrow therapeutic window due to cytokine release syndrome (CRS) induced toxicity, limited number of functional T cells, and an immunosuppressive tumor microenvironment (TME). Engagement of CD28 co-stimulatory domain can promote T cell fitness and sustained activation to overcome T -cell anergy. Here, we describe the development of a novel tri-specific antibody (TsAb) platform which is designed to optimally engage T cells via co-stimulation of both CD3 and of CD28 to achieve more effective tumor cell killing without increased CRS induction. To that end, we identified three selected formats (TsAb-1, TsAb-2 and TsAb-3) based on improved properties of the antibody formats, such as safety, efficacy, stability and developability. These formats have been developed to target three different well-established solid tumor-associated antigens (TAAs) as proof of concept. The binding activity of TsAbs to TAA, CD3 and CD28 was evaluated by ELISA and flow cytometry. Tumor cell cytotoxicity was assessed and secretion of cytokines by immune cells using a tumor cell-PBMC co-culture assay. Effective engagement of CD3 and CD28 was evaluated by flow analysis of CD25 Bcl-xL expression, respectively. Additionally, flow immunophenotyping was performed to characterize the differentiation of memory T cell subsets. Our results show that while all three TsAb formats displayed comparable cytotoxicity, TsAb-3 triggered immune cell activation only upon TAA engagement and T cell synapse formation. TsAb-3 showed > 90% purity, no CRS induction in PBMCs alone or in coculture with low TAA-expressing tumor cells. Additionally, tethered TsAb-3 format was unable to stimulate PBMCs in the absence of TAA. Upon further analysis of T cell phenotype differentiation, TsAb-3 showed enhanced CD25+ T cell activation, Bcl-xL induction and increased T-effector and memory cell phenotypes on day 3 and day 7. In conclusion, our data support further development of TsAb as a versatile and improved T cell engager therapeutics for the treatment of solid malignancies. These versatile formats can be utilized not only in T cell engager therapeutics, but also in rationale combinations with immune check point inhibitors. Citation Format: Reshmi Nair, Veena Somasundaram, Arvind Goswami, Jaya Bhatnagar, Sourav Paul, Chaitali Dey, Kalyanaraman Vaidyanathan, Rajkumar Paul, Sankar Periasamy, Mary Helen KG, Mahalakshmi NV, Abhishek Sinha, Rachel Salazar, Avanish K. Varshney. An improved trispecific antibody (TsAb) platform for optimally engaging T cells for the treatment of solid tumor malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1864.

JAK-STAT signalling shapes the NF-κB response in CLL towards venetoclax sensitivity or resistance via Bcl-XL.

Preventing or overcoming resistance to the Bcl-2 inhibitor venetoclax is an emerging unmet clinical need in patients with chronic lymphocytic leukaemia (CLL). The upregulation of anti-apoptotic Bcl-2 members through signalling pathways within the tumor microenvironment appears as a major factor leading to resistance to venetoclax. Previously, we reported that T cells can drive resistance through CD40 and non-canonical NF-κB activation and subsequent Bcl-XL induction. Moreover, the T cell-derived cytokines IL-21 and IL-4 differentially affect Bcl-XL expression and sensitivity to venetoclax via unknown mechanisms. Here, we mechanistically dissected how Bcl-XL is regulated in the context of JAK-STAT signalling in primary CLL. First, we demonstrated a clear antagonistic role of IL-21/STAT3 signalling in the NF-κB-mediated expression of Bcl-XL, whereas IL-4/STAT6 further promoted the expression of Bcl-XL. In comparison, Bfl-1, another NF-κB target, was not differentially affected by either cytokine. Second, STAT3 and STAT6 affected Bcl-XL transcription by binding to its promoter without disrupting the DNA-binding activity of NF-κB. Third, in situ proximity ligation assays (isPLAs) indicated crosstalk between JAK-STAT signalling and NF-κB, in which STAT3 inhibited canonical NF-κB by accelerating nuclear export, and STAT6 promoted non-canonical NF-κB. Finally, NF-κB inducing kinase (NIK) inhibition interrupted the NF-κB/STAT crosstalk and resensitized CLL cells to venetoclax. In conclusion, we uncovered distinct crosstalk mechanisms that shape the NF-κB response in CLL towards venetoclax sensitivity or resistance via Bcl-XL, thereby revealing new potential therapeutic targets.

Wnt/β-Catenin Protects Lymphocytes from HIV-Mediated Apoptosis via Induction of Bcl-xL.

HIV infection mediates the apoptosis of lymphocytes, the mechanism of which is multifaceted. Here, we evaluated the role of Wnt/β-catenin signaling in HIV-associated T cell apoptosis, as Wnt/β-catenin regulates the transcriptional activity of genes impacting apoptosis. We specifically investigated the role of the Wnt/β-catenin pathway in the HIV-associated apoptosis of CD4+ T cells and CD4dimCD8bright T cells, a population that is infected by HIV. We found that the induction of β-catenin, via a 6-bromoindirubin-3-oxime (BIO), significantly rescued HIV-infected CD4+ and CD4dimCD8bright T cells from apoptosis by >40–50%. Further, a small-molecule inhibitor of the Wnt/β-catenin pathway (PNU-74654) reversed BIO-mediated protection from HIV-associated apoptosis. BIO also induced Bcl-xL, an anti-apoptotic protein, and a target gene of Wnt/β-catenin, in CD4+ and CD4dimCD8bright T cells by approximately 3-fold. Inhibiting Bcl-xL by WEHI-539 abrogated β-catenin-mediated apoptotic protection in infected CD4+ and CD4dimCD8bright T cells. Collectively, these findings demonstrate that engaging Wnt/β-catenin signaling in HIV-infected T cells protects them from HIV-associated apoptosis by inducing Bcl-xL.

Aryl Hydrocarbon Receptor Promotes Cell Growth, Stemness Like Characteristics, and Metastasis in Human Ovarian Cancer via Activation of PI3K/Akt, β-Catenin, and Epithelial to Mesenchymal Transition Pathways.

Ovarian cancer (OC) ranks first in cancer-related deaths out of all female reproductive malignancies with high-pitched tumor relapse and chemoresistance. Several reports correlate cancer occurrences with exposure to xenobiotics via induction of a protein receptor named aryl hydrocarbon receptor (AhR). However, the effect of AhR on OC proliferation, expansion, and chemoresistance remains unrevealed. For this purpose, OC cells A2780 and A2780cis cells were treated with AhR activator, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the effects were determined by Real-Time Cell Analyzer, clonogenic assay, flow cytometry, immunoblotting and wound healing assay. Our results showed that activation of AhR by TCDD in A2780 cells induced the PI3K/AKT pathway followed by induction of anti-apoptotic proteins BCL-2, BCL-xl, and MCL-1. In addition, a significant increase in stemness marker aldehyde dehydrogenase (ALDH1) was observed. This effect was also associated with an accumulation of β-catenin, a Wnt transcription factor. Moreover, we observed induction of epithelial to mesenchymal transition (EMT) upon AhR activation. In conclusion, the results from the current study confirm that AhR mediates OC progression, stemness characteristics, and metastatic potential via activation of PI3K/Akt, Wnt/β-catenin, and EMT. This study provides a better insight into the modulatory role of AhR that might help in developing novel therapeutic strategies for OC treatment.

Suppression of non-small-cell lung cancer A549 tumor growthby an mtDNA mutation-targeting pyrrole-imidazole polyamide-triphenylphosphonium and a senolytic drug.

Certain somatic mutations in mtDNA were associated with tumor progression and frequently found in a homoplasmic state. We recently reported that pyrrole‐imidazole polyamide conjugated with the mitochondria‐delivering moiety triphenylphosphonium (PIP‐TPP) targeting an mtDNA mutation efficiently induced apoptosis in cancer cells with the mutation but not normal cells. Here, we synthesized the novel PIP‐TPP, CCC‐021‐TPP, targeting ND6 14582A > G homoplasmic missense mutation that is suggested to enhance metastasis of non‐small‐cell lung cancer A549 cells. CCC‐021‐TPP did not induce apoptosis but caused cellular senescence in the cells, accompanied by a significant induction of antiapoptotic BCL‐XL. Simultaneous treatment of A549 cells with CCC‐021‐TPP and the BCL‐XL selective inhibitor A‐1155463 resulted in apoptosis induction. Importantly, the combination induced apoptosis and suppressed tumor growth in an A549 xenografted model. These results highlight the potential of anticancer therapy with PIP‐TPPs targeting mtDNA mutations to induce cell death even in apoptosis‐resistant cancer cells when combined with senolytics.

Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL

AbstractChronic lymphocytic leukemia (CLL) cells cycle between lymph node (LN) and peripheral blood (PB) and display major shifts in Bcl-2 family members between those compartments. Specifically, Bcl-XL and Mcl-1, which are not targeted by the Bcl-2 inhibitor venetoclax, are increased in the LN. Because ibrutinib forces CLL cells out of the LN, we hypothesized that ibrutinib may thereby affect expression of Bcl-XL and Mcl-1 and sensitize CLL cells to venetoclax. We investigated expression of Bcl-2 family members in patients under ibrutinib or venetoclax treatment, combined with dissecting functional interactions of Bcl-2 family members, in an in vitro model of venetoclax resistance. In the PB, recent LN emigrants had higher Bcl-XL and Mcl-1 expression than did cells immigrating back to the LN. Under ibrutinib treatment, this distinction collapsed; significantly, the pretreatment profile reappeared in patients who relapsed on ibrutinib. However, in response to venetoclax, Bcl-2 members displayed an early increase, underlining the different modes of action of these 2 drugs. Profiling by BH3 mimetics was performed in CLL cells fully resistant to venetoclax due to CD40-mediated induction of Bcl-XL, Mcl-1, and Bfl-1. Several dual or triple combinations of BH3 mimetics were highly synergistic in restoring killing of CLL cells. Lastly, we demonstrated that proapoptotic Bim interacts with antiapoptotic Bcl-2 members in a sequential manner: Bcl-2 > Bcl-XL > Mcl-1 > Bfl-1. Combined, the data indicate that Bcl-XL is more important in venetoclax resistance than is Mcl-1 and provide biological rationale for potential synergy between ibrutinib and venetoclax.

Pharmacological inhibition of androgen receptor expression induces cell death in prostate cancer cells.

The androgen receptor (AR) plays an important role in the pathogenesis and development of prostate cancer (PCa). Mostly, PCa progresses to androgen-independent PCa, which has activated AR signaling from androgen-dependent PCa. Thus, inhibition of AR signaling may be an important therapeutic target in androgen-dependent and castration-resistant PCa. In this study, we determined the anticancer effect of a newly found natural compound, sakurasosaponin (S-saponin), using androgen-dependent and castration-resistant PCa cell lines. S-saponin induces mitochondrial-mediated cell death in both androgen-dependent (LNCaP) and castration-resistant (22Rv1 and C4-2) PCa cells, via AR expression. S-saponin treatment induces a decrease in AR expression in a time- and dose-dependent manner and a potent decrease in the expression of its target genes, including prostate-specific antigen (PSA), transmembrane protease, serin 2 (TMPRSS2), and NK3 homeobox 1 (NKX3.1). Furthermore, S-saponin treatment decreases B-cell lymphoma-extra large (Bcl-xL) and mitochondrial membrane potential, thereby increasing the release of cytochrome c into the cytosol. Moreover, Bcl-xL inhibition and subsequent mitochondria-mediated cell death caused by S-saponin were reversed by Bcl-xL or AR overexpression. Interestingly, S-saponin-mediated cell death was significantly reduced by a reactive oxygen species (ROS) scavenger, N-acetylcystein. Animal xenograft experiments showed that S-saponin treatment significantly reduced tumor growth of AR-positive 22Rv1 xenografts but not AR-negative PC-3 xenografts. Taken together, for the first time, our results revealed that S-saponin induces mitochondrial-mediated cell death in androgen-dependent and castration-resistant cells through regulation of AR mechanisms, including downregulation of Bcl-xL expression and induction of ROS stress by decreasing mitochondrial membrane potential.

PTP\u03c3 inhibitors promote hematopoietic stem cell regeneration

Receptor type protein tyrosine phosphatase-sigma (PTPσ) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPσ is also expressed by hematopoietic stem cells (HSCs). Here, we describe small molecule inhibitors of PTPσ that promote HSC regeneration in vivo. Systemic administration of the PTPσ inhibitor, DJ001, or its analog, to irradiated mice promotes HSC regeneration, accelerates hematologic recovery, and improves survival. Similarly, DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPσ and antagonizes PTPσ via unique non-competitive, allosteric binding. Mechanistically, DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-XL. Furthermore, treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective, small-molecule PTPσ inhibitors for human hematopoietic regeneration.

Epigenetic treatment-mediated modulation of PD-L1 predicts potential therapy resistance over response markers in myeloid malignancies: A molecular mechanism involving effectors of PD-L1 reverse signaling.

Recent evidence suggests an association exists between resistance to epigenetic therapy (EGT) and the expression of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) in myeloid malignancies. Biomarkers are required to predict resistance to EGT in myeloid malignancies, which together with the delineation of associated molecular mechanisms, may provide additional understanding for novel treatment strategies when investigating resistance to EGT. The present study aimed to investigate the in vivo effects of EGT on the expression of PD-1, PD-L1 and orphan nuclear receptor NUR77 with clinical responses in patients with myeloid malignancies. In addition, in vitro and in vivo characterization of the effects of EGT on the expression of NF-κB and Bcl-xL, potential downstream targets of PD-L1 reverse signaling, were evaluated to determine components of the molecular mechanism responsible for these effects. The in vivo effects of EGT on the expression of PD-1, PD-L1 and a previously identified molecular marker of response to EGT, NUR77 was characterized in peripheral blood mononuclear cells (PBMC) from patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) treated with either azacytidine (Aza) alone or a combination of Aza and the histone deacetylase inhibitor (HDACi) LBH-589 during the first cycle of therapy. The correlation of clinical responses to treatment with EGT with the expression of PD-1, PD-L1 and NUR77 demonstrated that the induction of PD-L1 mRNA levels was associated with resistance to EGT despite the concurrent augmentation of NUR77 expression. The characterization of potential downstream effector molecules of reverse PD-L1 signaling identified EGT-mediated induction of Bcl-xL and NF-κB mRNA expression in vitro and in vivo, suggesting a potential anti-apoptotic molecular mechanism was responsible for PD-L1-mediated resistance to EGT. Taken together, these observations suggest that enhanced PD-L1 expression may confer resistance to EGT over known EGT response markers in myeloid malignancies, and provides a potential molecular mechanism involving the modulation of effectors of PD-L1 reverse signaling, which may in-part, be responsible for these effects.

IL-21 and anti-CD40 restore Bcl-2 family protein imbalance in vitro in low-survival CD27+ B cells from CVID patients

Common variable immunodeficiency (CVID) is characterized by an abnormal B cell differentiation to memory and antibody-secreting B cells. The defective functionality of CVID patients’ B cells could be the consequence of alterations in apoptosis regulation. We studied the balance of Bcl-2 family anti-/pro-apoptotic proteins to identify molecular mechanisms that could underlie B cell survival defects in CVID. We used flow cytometry to investigate Bcl-2, Bcl-XL, Bax, and Bim expression in B cells ex vivo and after anti-CD40 or anti-BCR activation with or without IL-21, besides to spontaneous and stimulation-induced Caspase-3 activation and viable/apoptotic B cell subpopulations. We found increased basal levels of Bax and Bim in CVID B cells that correlated with low viability and high Caspase-3 activation only in CD27+ B cells, particularly in a subgroup of apoptosis-prone CVID (AP-CVID) patients with low peripheral B cell counts and high autoimmunity prevalence (mostly cytopenias). We detected a broad B cell defect in CVID regarding Bcl-2 and Bcl-XL induction, irrespective of the stimulus used. Therefore, peripheral CVID memory B cells are prompted to die from apoptosis due to a constitutive Bcl-2 family protein imbalance and defective protection from activation-induced apoptosis. Interestingly, anti-CD40 and IL-21 induced normal and even higher levels of Bcl-XL, respectively, in CD27+ B cells from AP-CVID, which was accompanied by cell viability increase. Thus low-survival memory B cells from AP-CVID can overcome their cell death regulation defects through pro-survival signals provided by T cells. In conclusion, we identify apoptosis regulation defects as disease-contributing factors in CVID. B cell counts and case history of cytopenias might be useful to predict positive responses to therapeutic approaches targeting T-dependent signaling pathways.

Rational targeted therapies to overcome microenvironment-dependent expansion of mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) accumulates in lymphoid organs, but disseminates early on in extranodal tissues. Although proliferation remains located in lymphoid organs only, suggesting a major role of the tumor ecosystem, few studies have assessed MCL microenvironment. We therefore cocultured primary circulating MCL cells from 21 patients several weeks ex vivo with stromal or lymphoid-like (CD40L) cells to determine which interactions could support their proliferation. We showed that coculture with lymphoid-like cells, but not stromal cells, induced cell-cycle progression, which was amplified by MCL-specific cytokines (insulin-like growth factor-1, B-cell activating factor, interleukin-6, interleukin-10). Of interest, we showed that our model recapitulated the MCL in situ molecular signatures (ie, proliferation, NF-κB, and survival signatures). We further demonstrated that proliferating MCL harbored an imbalance in Bcl-2 family expression, leading to a consequent loss of mitochondrial priming. Of interest, this loss of priming was overcome by the type II anti-CD20 antibody obinutuzumab, which counteracted Bcl-xL induction through NF-κB inhibition. Finally, we showed that the mitochondrial priming directly correlated with the sensitivity toward venetoclax and alkylating drugs. By identifying the microenvironment as the major support for proliferation and drug resistance in MCL, our results highlight a selective approach to target the lymphoma niche.

Bcl-2 Members As Drug Target and Biomarkers for Response to Ibrutinib and Venetoclax in CLL

Introduction.BCL-2 family members are crucial determinants for survival in normal and malignant B cells. Venetoclax, the BCL-2 targeting drug, was recently clinically approved for CLL. CLL cells cycle between the lymph node (LN) and peripheral blood, and between those compartments display large changes in BCL-XL and MCL-1, which are not targeted by Venetoclax. However, novel BH3 mimetics specific for BCL-XL and MCL-1 are under preclinical development. Ibrutinib is a clinically successful BTK inhibitor that forces Chronic Lymphocytic Leukemia (CLL) cells out of the lymph node. After prolonged application resistance develops in a fraction of patients, who then show fast disease progression. Thus, both Venetoclax and Ibrutinib have potential drawbacks when applied as single agents. For long-term successful application of new drug combinations, it is crucial to understand BCL-2 member functionality of malignant B cells in relation to their normal counterparts. In parallel, we studied clinical samples of CLL patients under Ibrutinib or Venetoclax treatment for changes in BCL-2 family members.Experimental procedures.FACS sorting of tonsil B subsets, mRNA and protein profiling, co-culture of CLL cells, co-IP of BCL-2 members, cell death assays with BH3 mimetics specific for BCL-2, BCL-XL or MCL-1 (ABT-199, WEHI-539, A-1210477), intracellular FACS for BCL-2, BCL-XL and MCL-1 in CLL (CD19/CD5/CXCR4).Results.We mapped the clearly distinct expression profiles for BCL-2 members in tonsillar naïve, germinal center (GC), plasma (PC) and memory B cells, which translate into different BH3 mimetic sensitivity profiles. In brief, naïve and memory cells rely on BCL-2, GC cells on MCL-1, and PC on BCL-XL. These approaches were extended to primary CLL cells. In a LN model where cells are fully resistant to single agent ABT-199 due to induction of MCL-1, BCL-XL and BFL-1, we find that dual or triple BH3 mimetic combinations restore killing. Moreover, there is differential sensitivity to (combinations of) BH3 mimetics between healthy and malignant B cells, suggesting there is a therapeutic window.Furthermore, intracellular FACS staining for BCL-2, MCL-1, and BCL-XL was performed on patients treated with Ibrutinib or Venetoclax. In untreated patients, recent LN emigrants (CD5hi/CXCR4lo) have higher BCL-XL and MCL-1 expression than 'old' returning cells (CD5lo/CXCR4hi). This distinction collapses under Ibrutinib treatment, demonstrating that Ibrutinib affects pro-survival BCL-2 members in vivo. Interestingly, in patients that develop Ibrutinib resistance the MCL-1 or BCL-XL pre-treatment profile reappears. In contrast, under Venetoclax treatment BCL-2 member levels do not change and can even increase in some patients, underlining the different mode of action of the two drugs. The pattern differed among patients (n=5) and increases were observed for BCL-2, BCL-XL, or MCL-1.Conclusions. BH3 mimetics have not only found their way to the clinic, but can also be used as tools for BCL-2 functionality profiling. Secondly, our ex vivo data underline that combination treatment between (paired) BH3 mimetics and/or Ibrutinib may afford better long-term efficacy in CLL than single agents. Thirdly, BCL-2 members may be useful as biomarkers for response and progression. DisclosuresEldering:Roche: Research Funding; Gilead: Research Funding. Brown:Pharmacyclics: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Genetech: Consultancy. Kater:Celgene: Research Funding.

Calcineurin inhibitors cyclosporin A and tacrolimus protect against podocyte injury induced by puromycin aminonucleoside in rodent models.

Podocyte injury and the appearance of proteinuria are features of minimal-change disease (MCD). Cyclosporin A (CsA) and tacrolimus (FK506) has been reported to reduce proteinuria in patients with nephrotic syndrome, but mechanisms remain unknown. We, therefore, investigated the protective mechanisms of CsA and FK506 on proteinuria in a rat model of MCD induced by puromycin aminonucleoside (PAN) and in vitro cultured mouse podocytes. Our results showed that CsA and FK506 treatment decreased proteinuria via a mechanism associated to a reduction in the foot-process fusion and desmin, and a recovery of synaptopodin and podocin. In PAN-treated mouse podocytes, pre-incubation with CsA and FK506 restored the distribution of the actin cytoskeleton, increased the expression of synaptopodin and podocin, improved podocyte viability, and reduced the migrating activities of podocytes. Treatment with CsA and FK506 also inhibited PAN-induced podocytes apoptosis, which was associated with the induction of Bcl-xL and inhibition of Bax, cleaved caspase 3, and cleaved PARP expression. Further studies revealed that CsA and FK506 inhibited PAN-induced p38 and JNK signaling, thereby protecting podocytes from PAN-induced injury. In conclusion, CsA and FK506 inhibit proteinuria by protecting against PAN-induced podocyte injury, which may be associated with inhibition of the MAPK signaling pathway.

Disturbed canonical nuclear factor of κ light chain signaling in B cells of patients with common variable immunodeficiency

Most patients with common variable immunodeficiency (CVID) present with severely reduced switched memory B-cell counts, and some display an increase of CD21low B-cell counts (CVID 21low), whereas others do not (CVID 21norm). Altered B-cell receptor (BCR) signaling might contribute to the defective memory formation observed in patients with CVID. We sought to investigate canonical nuclear factor of κ light chain (NF-κB) signaling in Bcells from patients with CVID as a central pathway in B-cell differentiation. Degradation of inhibitor of κBα (IκBα) and p65 phosphorylation, nuclear translocation of p65, and regulation of target genes and cell function were investigated after different modes of B-cell stimulation. BCR-mediated canonical NF-κB signaling was impaired in all mature naive CVID-derived Bcells. This impairment was more profound in naive Bcells from CVID 21low patients than CVID 21norm patients and most pronounced in CD21low Bcells. The signaling defect translated into reduced induction of Bcl-xL and IκBα, 2 bona fide target genes of the canonical NF-κB pathway. CD40 ligand- and Toll-like receptor 9-mediated signaling were less strongly altered. Signaling in CD21low Bcells but not CD21+ Bcells of patients with HIV was similarly affected. Combined with the previous description of disturbed Ca2+ signaling, the discovery of NF-κB signaling defects, especially in CVID 21low patients, suggests a broad underlying signaling defect affecting especially BCR-derived signals. Given the immune phenotype of monogenic defects affecting Ca2+ and NF-κB signaling, the latter is more likely to contribute to the humoral deficiency. The strongly disturbed BCR signaling of CD21low Bcells is characteristic for this cell type and independent of the underlying disease.

PACS-2 mediates the ATM and NF-κB-dependent induction of anti-apoptotic Bcl-xL in response to DNA damage.

Nuclear factor kappa B (NF-κB) promotes cell survival in response to genotoxic stress by inducing the expression of anti-apoptotic proteins including Bcl-xL, which protects mitochondria from stress-induced mitochondrial outer membrane permeabilization (MOMP). Here we show that the multifunctional sorting protein Pacs-2 (phosphofurin acidic cluster sorting protein-2) is required for Bcl-xL induction following DNA damage in primary mouse thymocytes. Consequently, in response to DNA damage, Pacs-2(-/-) thymocytes exhibit a blunted induction of Bcl-xL, increased MOMP and accelerated apoptosis. Biochemical studies show that cytoplasmic PACS-2 promotes this DNA damage-induced anti-apoptotic pathway by interacting with ataxia telangiectasia mutated (ATM) to drive NF-κB activation and induction of Bcl-xL. However, Pacs-2 was not required for tumor necrosis factor-α-induced NF-κB activation, suggesting a role for PACS-2 selectively in NF-κB activation in response to DNA damage. These findings identify PACS-2 as an in vivo mediator of the ATM and NF-κB-dependent induction of Bcl-xL that promotes cell survival in response to DNA damage.

Cycling hypoxia induces chemoresistance through the activation of reactive oxygen species-mediated B-cell lymphoma extra-long pathway in glioblastoma multiforme

BackgroundCycling hypoxia is a well-recognized phenomenon within animal and human solid tumors. It contributes to the resistance to cytotoxic therapies through anti-apoptotic effects. However, the mechanism underlying cycling hypoxia-mediated anti-apoptosis remains unclear.MethodsReactive oxygen species (ROS) production, activation of the hypoxia-inducible factor-1 alpha (HIF-1α) and nuclear factor-κB (NF-κB) signaling pathways, B-cell lymphoma extra-long (Bcl-xL) expression, caspase activation, and apoptosis in in vitro hypoxic stress-treated glioblastoma cells or tumor hypoxic cells derived from human glioblastoma xenografts were determined by in vitro ROS analysis, reporter assay, western blotting analysis, quantitative real-time PCR, caspase-3 activity assay, and annexin V staining assay, respectively. Tempol, a membrane-permeable radical scavenger, Bcl-xL knockdown, and specific inhibitors of HIF-1α and NF-κB were utilized to explore the mechanisms of cycling hypoxia-mediated resistance to temozolomide (TMZ) in vitro and in vivo and to identify potential therapeutic targets.ResultsBcl-xL expression and anti-apoptotic effects were upregulated under cycling hypoxia in glioblastoma cells concomitantly with decreased responses to TMZ through ROS-mediated HIF-1α and NF-κB activation. Tempol, YC-1 (HIF-1 inhibitor), and Bay 11-7082 (NF-κB inhibitor) suppressed the cycling hypoxia-mediated Bcl-xL induction in vitro and in vivo. Bcl-xL knockdown and Tempol treatment inhibited cycling hypoxia-induced chemoresistance. Moreover, Tempol treatment of intracerebral glioblastoma-bearing mice combined with TMZ chemotherapy synergistically suppressed tumor growth and increased survival rate.ConclusionsCycling hypoxia-induced Bcl-xL expression via ROS-mediated HIF-1α and NF-κB activation plays an important role in the tumor microenvironment-promoted anti-apoptosis and chemoresistance in glioblastoma. Thus, ROS blockage may be an attractive therapeutic strategy for tumor microenvironment-induced chemoresistance.

Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström macroglobulinemia.

Waldenström Macroglobulinemia (WM) is a proliferative disorder of IgM secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.

49 Reversal of Mutant KRAS-Mediated Apoptosis Resistance by Concurrent Bcl-xL Antagonism and Induction of Pro-Apoptotic BH3-Only Proteins in Colorectal Carcinoma Cells

Background. KRAS mutations, detected in at least 40% of human colorectal cancers (CRC), contribute to de novo apoptosis resistance and ultimately treatment failure. Aims. To study the mechanism of KRAS-mediated apoptosis resistance, we examined proand anti-apoptotic Bcl-2 family proteins in isogenic mutant vs wild type KRAS colorectal cancer (CRC) cells. Experimental Methods. Isogenic KRAS mutant CRC cells (HCT116, DLD1) and KRAS mutant SW620 cells were analyzed for Bcl-2 family proteins by immunoblotting. Cells were incubated with ABT-263 (1 μM), carfilzomib (0-50nM) or their combination; apoptosis was measured by annexin V+ labeling and caspase cleavage. Drug synergy was determined by calculation of a combination index. Protein-protein interaction was examined by immunoprecipitation. Knockdown of ERK or KRAS was achieved by siRNA. Ectopic expression of mutant KRAS or Noxa was achieved by a constitutive or doxycyline-inducible expression system, respectively. Results. We found that mutant KRAS cells showed upregulation of Bcl-xL that was attenuated by KRAS siRNA or reproduced by ectopic expression of mutant KRAS. Upregulation of Bcl-xL by mutant KRAS was mediated by ERK shown using EKR siRNA. Bcl-xL upregulation occurred at the level of mRNA and protein shown using actinomycin D and cyclohexamide, respectively. While BH3 mimetic agents can antagonize Bcl-2/BclxL, they are generally insufficient to overcome apoptosis resistance in solid tumors. Therefore, we evaluated a strategy of concurrent Bcl-xL antagonism and induction of pro-apoptotic BH3-only proteins. Carfilzomib, an irreversible proteasome inhibitor, was shown to induce Noxa and Bik proteins but failed to reverse mutant KRAS-mediated apoptosis resistance. However, concurrent antagonism of Bcl-xL by the BH3 mimetic, ABT-263, combined with carfilzomib was shown to synergistically enhance apoptosis in isogenic mutant KRAS CRC cell lines (Figure). In support of this strategy, we mimicked Noxa induction, seen with carfilzomib, using a doxycycline-inducible expression system. Ectopic Noxa was shown to markedly enhance apoptosis by ABT-263. A mechanism for drug synergy was suggested by the finding that Noxa and Bik proteins can sequester anti-apoptotic Mcl-1, while ABT-263 was able to release Bik and Bak from binding to Bcl-xL. Conclusion. Our novel findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance. Antagonism of Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. Together, these preclinical data suggest a promising therapeutic strategy to overcome apoptosis resistance in KRAS mutant CRC cells.

Metformin protects primary rat hepatocytes against oxidative stress-induced apoptosis.

The majority of chronic liver diseases are accompanied by oxidative stress, which induces apoptosis in hepatocytes and liver injury. Recent studies suggest that oxidative stress and insulin resistance are important in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the pathophysiology of diabetes complications. Metformin has been shown to be hepatoprotective in the insulin-resistant and leptin-deficient ob/ob mouse model of NAFLD. However, the mechanism involved in the protective effects of metformin has not been elucidated yet. Therefore, we investigated the protective effect of metformin against oxidative stress-induced apoptosis. Primary rat hepatocytes were exposed to the oxidative stress-generating compound menadione in the presence and absence of metformin. Apoptosis was determined by measuring caspase activity and poly(ADP-ribose) polymerase (PARP)-cleavage, and necrosis was measured by Sytox Green nuclear staining. We demonstrate that (1) Metformin inhibits menadione-induced caspase-9,-6,-3 activation and PARP-cleavage in a concentration-dependent manner. (2) Metformin increases menadione-induced heme oxygenase-1 (HO-1) expression and inhibits c-Jun N-terminal kinase (JNK)-phosphorylation. (3) Metformin does not induce necrosis in primary hepatocytes. Metformin protects hepatocytes against oxidative stress-induced caspase activation, PARP-cleavage and apoptosis. The anti-apoptotic effect of metformin is in part dependent on HO-1 and bcl-xl induction and inhibition of JNK activation and independent of insulin signaling. Our results elucidate novel protective mechanisms of metformin and indicate that metformin could be investigated as a novel therapeutic agent for the treatment of oxidative stress-related liver diseases.