B-cell Apoptosis Research Articles

Genetic predisposition to B-cell acute lymphoblastic leukemia at 14q11.2 is mediated by a CEBPE promoter polymorphism

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Genome-wide association studies have shown variation at 14q11.2 influences ALL risk. We sought to decipher causal variant(s) at 14q11.2 and the mechanism of tumorigenesis. We show rs2239630 G>A resides in the promoter of the CCAT enhancer-binding protein epsilon (CEBPE) gene. The rs2239630-A risk allele is associated with increased promotor activity and CEBPE expression. Depletion of CEBPE in ALL cells reduces cell growth, correspondingly CEBPE binds to the promoters of electron transport and energy generation genes. RNA-seq in CEBPE depleted cells demonstrates CEBPE regulates the expression of genes involved in B-cell development (IL7R), apoptosis (BCL2), and methotrexate resistance (RASS4L). CEBPE regulated genes significantly overlapped in CEBPE depleted cells, ALL blasts and IGH-CEBPE translocated ALL. This suggests CEBPE regulates a similar set of genes in each, consistent with a common biological mechanism of leukemogenesis for rs2239630 associated and CEBPE translocated ALL. Finally, we map IGH-CEBPE translocation breakpoints in two cases, implicating RAG recombinase activity in their formation.

PO-057 Genetic interaction and pathway based discovery of key regulators in multiple myeloma

IntroductionMultiple myeloma (MM) is a plasma cell dyscrasia characterised by proliferation of monoclonal plasma cells. As one of the most prevalent haematological malignancies, MM is genetically heterogeneous. Although 17 individual MM susceptibility loci have been identified, their inherent biological mechanism is sparsely understood. We carried out a polygenic interaction analysis with published whole-genome association studies (GWASs) to further characterise MM susceptibility with inflated genomic resolution in order to locate seemingly aberrant variations having temporal aggregation of risk.Material and methodsSingle nucleotide polymorphisms data from two MM GWAS representing 3999 cases and 7266 controls were subjected to genome-wide interaction analysis and followed-up with a meta-analysis. The identified loci were used for consequent gene set enrichment and tissue expression prioritisation to obtain functional evidence. Causal dependency between sentinel loci and functional variants was then interrogated with expression quantitative trait loci subject to summary data-based Mendelian randomization. Patient samples and relevant clinico-pathological information was obtained conditional on written informed consent and was subject to approval of corresponding ethical review boards at respective study centres in accordance with the tenets of Declaration of HelsinkiResults and discussionsA total of sixteen novel interacting pairs were identified along with indication of differential regulation in two principal signalling cascade families. Several previously identified and novel chromosomal regions influencing hematopoietic malignancies and contributing to pluripotent B-cell differentiation, survival, proliferation and apoptosis were identified. These loci were suggested to influence immune response and regulate immunoglobulin class-switch. Tissue/cell-type enrichment analysis showed that the genes associated with the identified loci were highly expressed in hemic-immune system tissue types and immune-related cell types.ConclusionOur polygenic interaction analysis renders novel insight into genetic susceptibility in MM. Related pathways provide some basic understanding of functional relation between discovered candidates as well as a biological basis of predisposition.

Transcriptome reveals B lymphocyte apoptosis in duck embryonic bursa of Fabricius mediated by mitochondrial and Fas signaling pathways

As a central immune organ unique to birds, the bursa of Fabricius (BF) provides a proper microenvironment for B-cell development. The bursal B-cells undergo rapid proliferation and differentiation at the embryonic stages, but 95% of them undergo apoptosis after hatching. Few studies have focused on the cause of bursal B-cells apoptosis at the embryonic stages in birds. To explore the cause, we compared the transcriptional profiles of three characteristic embryonic stages in duck, including embryonic day 14 (ED14), 22 (ED22) and 1 day after hatching (D1). Our results showed that the apoptotic B-cells were first observed at ED22 while there were no apoptotic B-cells at ED14. By performing enrichment analysis for DEGs and qRT-PCR, our results demonstrated that both mitochondrial and Fas signaling pathways mediated bursal B-cell apoptosis during the duck embryonic development. Further, protein-protein interactions (PPIs) and KEGG enrichment analysis together showed that BMP4, FoxO1 and IGF-1 may regulate bursal B-cells apoptosis. In addition, the DEGs showed two stage-specific expression patterns. By analyzing the genes of two expression patterns, the results indicated that B-cell false differentiation may be one of the reasons of apoptosis in the duck embryonic BF. Overall, these data demonstrated that from ED14-ED22, apoptosis of bursal B-cells was mediated by mitochondrial and Fas signaling pathways and could be regulated by BMP4, FoxO1 and IGF-1 in duck. One of the primary causes of bursal B-cell apoptosis may be false differentiation in B-cells.

Ibrutinib.

Abnormal B-cell receptor (BCR) signalling is a key mechanism of disease progression in B-cell malignancy. Bruton's tyrosine kinase (BTK) has a pivotal role in BCR signalling. Ibrutinib (PCI-32765) is a small molecule which serves as a covalent irreversible inhibitor of BTK. It is characterized by high selectivity for BTK and high potency. Ibrutinib is currently approved by the FDA and EMA for use in chronic lymphocytic leukaemia in any line of treatment, for treatment of Waldenstrom macroglobulinemia in patients who have received previous treatments or are not suitable to receive immunochemotherapy as well as for second line treatment of mantle cell lymphoma and for patients with marginal zone lymphoma who have received at least one prior anti-CD20-based therapy. In addition, there is emerging clinical data on its efficacy in ABC subtype diffuse large B-cell lymphoma, multiple myeloma and primary central nervous system lymphoma. Ibrutinib has opened new options for treatment of those patients that have relapsed or have been refractory to more classical modes of treatment. Moreover, Ibrutinib has been shown to be effective in patients that have been known to have little sensitivity to classical immunochemotherapy. Having a favourable risk profile, the substance is, unlike conventional immunochemotherapy, also suitable for the less physical fit patients. Cases of primary and secondary resistance to Ibrutinib have emerged and there is an ongoing effort to identify their mechanism and develop strategies to overcome them. Beyond its direct effects on survival and apoptosis of malignant B-cells, there is increasing evidence that Ibrutinib is able to modulate the tumour microenvironment to overcome mechanisms of immune evasion. This has sparked interest in use of the substance beyond lymphoid malignancy. This chapter discusses structure, mechanism of action and toxicities of Ibrutinib and also presents important preclinical and clinical data as well as mechanisms of Ibrutinib resistance. Combination strategies with immunotherapeutic strategies such as immune checkpoint blockade and CAR T-cell therapy may be synergistic and are currently under investigation.

Abstract 02: BCL-W significantly contributes to B-cell lymphoma survival and development

Abstract The ability of cells to undergo apoptosis in response to cellular stresses functions as a safeguard against tumorigenesis. Consequently, compromised apoptotic signaling must precede tumor development. To design effective therapies for cancer treatment, the underlying mechanisms that govern cell survival need to be understood. The anti-apoptotic proteins of the Bcl-2 family are key regulators of cell survival and are frequently overexpressed in malignancies, leading to increased cancer cell survival. Bcl-w, the closest anti-apoptotic relative to Bcl-2 and Bcl-xL, is essential for spermatogenesis, but was considered dispensable for other cell types. However, we recently uncovered a significant role for Bcl-w in B-cell survival and lymphomagenesis. Specifically, loss of Bcl-w conferred sensitivity to growth factor deprivation-induced B-cell apoptosis. In addition, apoptosis triggered by hyperproliferative signals from the Myc oncogene was accentuated by loss of Bcl-w. Consequences of elevated apoptosis were observed in vivo, as Bcl-w loss profoundly delayed Myc-mediated B-cell lymphoma development. We also determined Myc regulates Bcl-w expression through its transcriptional regulation of the miR-15 family. Importantly, evaluation of Burkitt lymphoma (BL) patient samples revealed BCL-W overexpression was highly selected for, and that targeting BCL-W using multiple methods induced apoptosis of BL cells. In addition, overexpression of BCL-W in BL cell lines conferred resistance to the BH3-mimetics ABT-737 and ABT-263 that inhibit anti-apoptotic BCL-2 family members. Furthermore, BCL-W was overexpressed in diffuse large B-cell lymphoma (DLBCL) and correlated with decreased patient survival. Analysis of multiple data sets of other aggressive and indolent B-cell lymphomas showed that BCL-W was frequently overexpressed in them as well. Collectively, our data indicate that BCL-W is an understudied contributor to B-cell lymphoma development and survival that appears to be as important as BCL-2 to B-cell lymphomas. In addition, BCL-W is a predictive biomarker that could improve prognostication and the development of more effective treatment strategies for B-cell lymphomas. Citation Format: Clare M. Adams, Ramkrishna Mitra, Jerald Z. Gong, Annette S. Kim, John K. Choi, Christine M. Eischen. BCL-W significantly contributes to B-cell lymphoma survival and development [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 02.

Role of Apoptosis in the Pathogenesis of Common Variable Immunodeficiency (CVID).

Common variable immunodeficiency (CVID) is a heterogeneous immune deficiency characterized by hypogammaglobulinemia. Since B cell maturation and differentiation is defective in this disorder, we evaluated apoptosis in B cells of patients with CVID compared with healthy donors (HD). Determination of peripheral blood B-cell subsets in CVID and HDs, was performed using flow cytometry. We compared total apoptosis, early apoptosis and late apoptosis/necrosis in unstimulated and stimulated B-cells of patients with CVID and HDs. We also assessed the expression of the anti-apoptotic molecule BCL2 mRNA levels in B-cells by real-time PCR in CVID patients compared with HDs. Total B-cell apoptosis was increased in both unstimulated and stimulated B-cells from CVID patients compared with HDs (p=0.02 and p=0.004). Early apoptosis in stimulated B-cells (p=0.04) and late apoptosis/necrosis of B-cells in both unstimulated and stimulated B-cells (p=0.04 and p=0.03, respectively) were significantly higher in CVID patients compared with HDs. There was a significant inverse correlation between the percentages of post germinal center B-cells in the peripheral blood of CVID patients compared with percentage of apoptotic B-cells. However, anti-apoptotic BCL2 expression was not significantly reduced in B-cells from CVID patients compared with HDs (p=0.16). Increased apoptosis of B-cells may be a factor in abnormality of differentiated B-cell subsets and the impaired endogenous immunoglobulin production in CVID patients. Further studies of the expression of pro/anti-apoptotic mediators in B-cells of CVID patients may shed light on the mechanism behind this increased B-cell apoptosis, and present potential therapeutic interventions in the future.

Role of purinergic signalling and proinflammatory cytokines in diabetes

Extracellular purines activate P1 adenosine receptors and P2 nucleotide receptors. These receptors are pre­sent on the pancreatic islet cells as well as on hepato­cytes, adipocytes, pancreatic blood vessels and nerves. ATP is released together with insulin from b-cell gran­ules in response to a rapid decrease in blood glucose levels. The ATP-dependent P2X receptor activation on pancreatic b-cells results in a positive autocrine signal and subsequent insulin secretion. Adenosine, through activation of P1 receptors present on adipocytes and pancreatic islet cells, inhibits the release of insulin. Adenosine activates A 2B receptors thereby stimulating production of IL-6 and other cytokines, which increases insulin resistance. Interleukin-6 also plays an important role in diabetes. In type 2 diabetes and obesity, the long-term increase of IL-6 concentration in blood above 5 pg/mL leads to the chronic and permanent increase in expression of SOCS3, contributing to the increase in insulin resistance in cells of the skeletal muscles, liver and adipose tissue. In diabetes there is an increased synthesis and release of pro-inflammatory cytokines, which cause the damage of the pancreatic islet cells, and in type 2 diabetes cause the development of insulin resistance. Ecto-enzymes metabolizing nucleotides are involved in the termination of the nucleotide signalling pathway and play the key role in regulation of extracel­lular ATP concentration. Ecto-NTPDases in cooperation with 5’-nucleotidase may significantly increase ecto-adenosine concentration. NTPDase3 activity has only been demonstrated on Langerhans cells. NTPDase3 may influence the secretion of insulin by hydrolysing adenine nucleotides. In diabetes the pro-inflammatory cytokines such as interleukin 1b (IL-1b), tumour ne­crosis factor-a (TNF-a) and interferon-g (IFN-g), as well as pancreatic derived factor PANDER are involved in the apoptosis of pancreatic b-cells. This causes distur­bance of the balance between pro-inflammatory and protective cytokines. We believe that neutralization of pro-inflammatory cytokines, especially interleukin 1b, with the IL-1 receptor antagonist (IL-1Ra) and/or IL-1b antibodies might cause the reduction of the inflamma­tory process in pancreas islets, normalize concentration of glucose in blood and decrease the insulin resistance. (Clin Diabetol 2017; 6, 3: 90–100)

A novel anti-CD22 scFv\u2013apoptin fusion protein induces apoptosis in malignant B-cells

CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv–apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv–apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.

Use of Anti-Thymocyte Globulin for Induction Therapy in Cardiac Transplantation: A Review

The most common causes of death after heart transplantation (HTx) include acute rejection and multi-organ failure in the early period and malignancy and cardiac allograft vasculopathy (CAV) in the late period. Polyclonal antibody preparations such as rabbit anti-thymocyte globulin (ATG) may reduce early acute rejection and the later occurrence of CAV after HTx. ATG therapy depletes T cells, modulates adhesion and cell-signaling molecules, interferes with dendritic cell function, and induces B-cell apoptosis and regulatory and natural killer T-cell expansion. Evidence from animal studies and from retrospective clinical studies in humans indicates that ATG can be used to delay calcineurin inhibitor (CNI) exposure after HTx, thus benefiting renal function, and to reduce the incidence of CAV and ischemia-reperfusion injury in the transplanted heart. ATG may reduce de novo antibody production after HTx. ATG does not appear to increase cytomegalovirus infection rates with longer prophylaxis (6–12 months). In addition, ATG may reduce the risk of lymphoproliferative disease and does not appear to confer an additive effect on acquiring lymphoma after HTx. Randomized, controlled trials may provide stronger evidence of ATG association with patient survival, graft rejection, renal protection through delayed CNI initiation, as well as other benefits. It can also help establish optimal dosing and patient criteria to maximize treatment benefits.

Abstract P1-02-10: Reduced serum B-cell maturation antigen levels predict poor outcome in metastatic breast cancer patients in a phase 3 randomized 2nd-line hormone therapy trial

Abstract Background: B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor family and has two ligands, B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL). These ligands activate cell proliferation and inhibit apoptosis of normal and malignant B-cells including in multiple myeloma (MM) cell lines. Berenson et al have recently reported that circulating BCMA levels are elevated in B-cell malignancies and can be used to monitor disease and predict PFS and OS for patients with MM, Waldenstroms's macroglobulinemia and chronic lymphocytic leukemia (CLL). On the other hand, recent studies have shown that serum BCMA levels are very low among patients with MM in complete remission with low antibody levels and those with primary immune deficiencies specifically those with combined variable immune deficiency and X-linked agammaglobulinemia. Studies of the potential role of serum BCMA for patients with solid tumors has not been evaluated to date. Methods: The pretreatment serum from 139 patients with hormone receptor-positive metastatic breast cancer who were enrolled in a phase 3 randomized clinical trial of second-line hormone therapy was evaluated using an ELISA for BCMA. The BCMA ELISA was from R&D Systems (Minneapolis, MN). Serum BCMA was correlated with TTP using categorical serum BCMA cutpoints. Results: Pretreatment serum BCMA levels had a median of 55.61 ng/ml, an interquartile range of 34.20 and 78.79 ng/ml, and full range from 3.99 to 1193.26 ng/ml. In univariate analysis for TTP, reduced serum BCMA correlated with shorter TTP at the following dichotomous cutpoints: 15 ng/ml [HR=2.60, p=0.064, n=6 (4.3%) of patients below cutpoint]; 20 ng/ml [HR=2.88, p=0.005, n=10 (7.2%) of patients below cutpoint]; 25 ng/ml [HR=2.16, p=0.023, n=13 (9.4%) of patients below cutpoint]; and 30 ng/ml [HR=1.77, p=0.016, n=27 (19.4%) of patients below the cutpoint]. Conclusions: In a phase 3 randomized clinical trial of second-line hormone therapy among patients with hormone receptor-positive metastatic breast cancer, reduced pretreatment serum BCMA was associated with shorter TTP. This may be due to the association of reduced serum BCMA with immune deficiency; and, thus, lead to shorter TTP among patients with metastatic breast cancer. Evaluation of serum BCMA as a new biomarker to predict outcomes for breast cancer and other solid tumor patients deserves further study. Citation Format: Ali SM, Leitzel K, Li M, Udd K, Wang J, Sanchez E, Chen H, Berenson J, Lipton A. Reduced serum B-cell maturation antigen levels predict poor outcome in metastatic breast cancer patients in a phase 3 randomized 2nd-line hormone therapy trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-10.

Vitiligo blood transcriptomics provides new insights into disease mechanisms and identifies potential novel therapeutic targets

BackgroundSignificant gaps remain regarding the pathomechanisms underlying the autoimmune response in vitiligo (VL), where the loss of self-tolerance leads to the targeted killing of melanocytes. Specifically, there is incomplete information regarding alterations in the systemic environment that are relevant to the disease state.MethodsWe undertook a genome-wide profiling approach to examine gene expression in the peripheral blood of VL patients and healthy controls in the context of our previously published VL-skin gene expression profile. We used several in silico bioinformatics-based analyses to provide new insights into disease mechanisms and suggest novel targets for future therapy.ResultsUnsupervised clustering methods of the VL-blood dataset demonstrate a “disease-state”-specific set of co-expressed genes. Ontology enrichment analysis of 99 differentially expressed genes (DEGs) uncovers a down-regulated immune/inflammatory response, B-Cell antigen receptor (BCR) pathways, apoptosis and catabolic processes in VL-blood. There is evidence for both type I and II interferon (IFN) playing a role in VL pathogenesis. We used interactome analysis to identify several key blood associated transcriptional factors (TFs) from within (STAT1, STAT6 and NF-kB), as well as “hidden” (CREB1, MYC, IRF4, IRF1, and TP53) from the dataset that potentially affect disease pathogenesis. The TFs overlap with our reported lesional-skin transcriptional circuitry, underscoring their potential importance to the disease. We also identify a shared VL-blood and -skin transcriptional “hot spot” that maps to chromosome 6, and includes three VL-blood dysregulated genes (PSMB8, PSMB9 and TAP1) described as potential VL-associated genetic susceptibility loci. Finally, we provide bioinformatics-based support for prioritizing dysregulated genes in VL-blood or skin as potential therapeutic targets.ConclusionsWe examined the VL-blood transcriptome in context with our (previously published) VL-skin transcriptional profile to address a major gap in knowledge regarding the systemic changes underlying skin-specific manifestation of vitiligo. Several transcriptional “hot spots” observed in both environments offer prioritized targets for identifying disease risk genes. Finally, within the transcriptional framework of VL, we identify five novel molecules (STAT1, PRKCD, PTPN6, MYC and FGFR2) that lend themselves to being targeted by drugs for future potential VL-therapy.

Genetic defects in PI3Kδ affect B-cell differentiation and maturation leading to hypogammaglobulineamia and recurrent infections

BackgroundMutations in PIK3CD and PIK3R1 cause activated PI3K-δ syndrome (APDS) by dysregulation of the PI3K-AKT pathway. MethodsWe studied precursor and peripheral B-cell differentiation and apoptosis via flowcytometry. Furthermore, we performed AKT-phosphorylation assays and somatic hypermutations (SHM) and class switch recombination (CSR) analysis. ResultsWe identified 13 patients of whom 3 had new mutations in PIK3CD or PIK3R1. Patients had low total B-cell numbers with increased frequencies of transitional B cells and plasmablasts, while the precursor B-cell compartment in bone marrow was relatively normal. Basal AKT phosphorylation was increased in lymphocytes from APDS patients and natural effector B cells where most affected. PI3K mutations resulted in altered SHM and CSR and increased apoptosis. ConclusionsThe B-cell compartment in APDS patients is affected by the mutations in PI3K. There is reduced differentiation beyond the transitional stage, increased AKT phosphorylation and increased apoptosis. This B-cell phenotype contributes to the clinical phenotype.

Sensitivity of Ibrutinib Exposed Chronic Lymphocytic Leukemia B-Cells to Inhibition of Axl Receptor Tyrosine Kinase

Introduction: The use of B-cell receptor (BCR) signal inhibitors-based therapies (e.g., Ibrutinib) for B-chronic lymphocytic leukemia (CLL) was initiated just a few years ago but has rapidly escalated due to their clinical efficacy and relative ease of use. However newer therapeutic approaches are needed due to multiple issues including the continued need to improve complete responses and reduce toxicity profiles. To that end our group has discovered a novel membrane target in the ubiquitous presence of Axl receptor tyrosine kinase (Axl RTK) on CLL B-cells and has reported that the Axl RTK inhibitor TP-0903 is able to induce apoptosis of CLL B-cells at nanomolar doses (Sinha, Clin Cancer Res, 2015). Given this we assessed if TP-0903 would be effective in the induction of apoptosis of leukemic B-cells from CLL patients who are currently on Ibrutinib therapy or whom have relapsed while on Ibrutinib treatment.Methods: Relapsed/refractory CLL patients (n=22) who were placed on Ibrutinib for progressive disease provided blood samples at a median of 3.2 months after Ibrutinib therapy initiation for these studies. We also obtained sequential samples on 8 patients from initial start of ibrutinib therapy and then over a 6 month follow-up period. CLL B-cells from these blood samples were subject to Ficoll separation, purified by using a Rosette Sep B-cell enrichment kit and then studied by flow cytometry to determine Axl RTK expression levels by flow cytometric analysis. Purified CLL B-cells (CD19+/CD5+) were cultured with TP-0903 in vitroat increasing doses (0.01µM - 0.50µM) for 24 hours and the LD50 dose was determined. In addition, 3 CLL patients who had been on Ibrutinib therapy and had a documented relapse were studied in similar fashion using TP-0903. LD50-sensitivity was measured. "LD50-sensitivity" was defined as an LD50 ≤0.50µM and "insensitive" was defined as an LD50 dose >0.50µM. CLL prognostic factors (e.g., FISH, IGHV mutation status, Rai stage, CD38, and CD49d) were evaluated at the time of ibrutinib treatment. Differences in factors between sensitive and insensitive cases were computed using the Kruskal-Wallis test for continuous variables and Chi-square test for categorical variables.Results: Twenty-two CLL patients (5 female, 17 male) were included in the analysis. Fourteen (64%) patients were found to be TP-0903 LD50-sensitive. Axl expression on CLL B-cells for this cohort was heterogeneous with a median of CD19+/CD5+ cells positive for Axl at 69.9% (range of 2.7-91.3%). The sensitive subjects tended to be younger with a median age at Ibrutinib treatment initiation of 62 vs 75.5 years (p=0.004). There were no significant differences in gender, FISH, IGHV mutation status, CD38, CD49d, or Rai stage between the sensitive and insensitive LD50 groups. There were no significant differences in relation to median Axl expression on CLL B-cells (sensitive: 72.6%, range: 2.7-91.3%; insensitive: 41.5%, range: 16.5-83.1%; p=0.35). The median number of treatments prior to initiation of ibrutinib did not differ between sensitivity groups (sensitive: 2.53, range: 8-10; insensitive: 43.5, range 12-20; p=0.2833). Association for ZAP70+ CLL B-cells tended to have more apoptosis induction by TP-0903 (sensitive: 84.6% ZAP70+; insensitive: 42.9% ZAP70+; p=0.052). In 8 CLL patients that were studied sequentially while on Ibrutinib continued to express Axl or increased their Axl expression (n=2) over a 3-6 month follow-up period. Three CLL patients who had relapsed on Ibrutinib were sensitive to TP-0903 with LD50 values of ≤0.50µM.Summary: Here we find that CLL B-cells from over 60% of relapsed CLL patients on Ibrutinib therapy were highly sensitive to the high-affinity Axl inhibitor TP-0903 with induction of apoptosis at nanomolar doses (≤0.50µM). The sensitivity of CLL B-cells to TP-0903 appears to be independent of Axl expression levels and of the known CLL prognostic factors but more evident for younger patients and for ZAP70+ expression status. Given this level of activity for apoptosis induction of CLL B-cells by TP-0903 encourages the further testing of this drug in clinical trials for CLL patients. DisclosuresParikh:Pharmacyclics: Honoraria, Research Funding. Shanafelt:Pharmacyclics: Research Funding; Janssen: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Hospira: Research Funding. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Pharmacyclics: Research Funding; Tolero Pharmaceuticals: Research Funding; Acerta: Research Funding; Gilead: Membership on an entity’s Board of Directors or advisory committees; Morpho-Sys: Membership on an entity’s Board of Directors or advisory committees; Infinity Pharm: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees.

HCV-ІНФЕКЦІЯ Й ЛІМФОПРОЛІФЕРАЦІЯ (ОГЛЯД ЛІТЕРАТУРИ)

У статті описано роль HCV-інфекції у розвитку лімфопроліферативних порушень. Представлено деякі ланки патогенезу позапечінкових проявів, таких, як аутоімунні порушення й неходжкінська лімфома (НХЛ), пов’язаних з HCV-інфекцією. Змішана кріоглобулінемія є найпоширенішим станом, пов’язаним з HCV-індукованою лімфопроліферацією і в деяких випадках здатна призвести до клінічних проявів, таких, як геморагічний васкуліт, пурпура, артралгії тощо, а також розвитку В-клітинної НХЛ. HCV-лімфогенез заснований на безперервній В-клітинній активації й інгібуванні В-клітинного апоптозу, що, імовірно, відіграє ключову роль у розвитку позапечінкових проявів, пов’язаних з HCV- інфекцією.

Endotoxemia contributes to CD27+ memory B-cell apoptosis via enhanced sensitivity to Fas ligation in patients with Cirrhosis.

Peripheral CD27+ memory B-cells become quantitatively reduced and dysfunctional in patients with cirrhosis through poorly characterized mechanisms. We hypothesized that the disappearance of CD27+ memory B-cells results from enhanced sensitivity to apoptosis caused by exposure to gut microbial translocation products. Using isolated naïve and memory B-cells from patients with cirrhosis and age-matched controls, ex vivo and activation-induced sensitivity to Fas-mediated apoptosis was assessed under relevant experimental conditions. We observed differential expression of CD95(Fas) in CD27+ B-cells from cirrhotic patients that was inversely correlated with peripheral CD27+ B-cell frequency. While memory B-cells from cirrhotic patients were resistant to Fas-mediated apoptosis ex vivo, Toll-like receptor 4(TLR4)-ligation restored Fas-sensitivity. Sensitivity to Fas-mediated apoptosis could be transferred to healthy donor memory B-cells by co-culturing these cells with plasma from cirrhotic patients, a sensitivity partially mediated by Fas and TLR4 signaling, and partially rescued via B-cell receptor crosslinking. We conclude that peripheral CD27+ memory B-cells in cirrhosis exhibit increased sensitivity to Fas-induced apoptosis in an activation-dependent manner to which endotoxin contributes, associated with reduced frequency of circulating memory B-cells. Destruction of this critical cell subset may contribute to the cirrhotic immunodeficiency state and heightened risk of systemic infections in advanced liver disease.

PI3K\u03b4 inhibition elicits anti-leukemic effects through Bim-dependent apoptosis

PI3Kδ plays pivotal roles in the maintenance, proliferation and survival of malignant B-lymphocytes. Although not curative, PI3Kδ inhibitors (PI3Kδi) demonstrate impressive clinical efficacy and, alongside other signaling inhibitors, are revolutionizing the treatment of hematological malignancies. However, only limited in vivo data are available regarding their mechanism of action. With the rising number of novel treatments, the challenge is to identify combinations that deliver curative regimes. A deeper understanding of the molecular mechanism is required to guide these selections. Currently, immunomodulation, inhibition of B-cell receptor signaling, chemokine/cytokine signaling and apoptosis represent potential therapeutic mechanisms for PI3Kδi. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced apoptosis in an in vivo model of chronic lymphocytic leukemia (CLL). In vitro, PI3Kδi-induced substantive apoptosis and disrupted microenvironment-derived signaling in murine (Eμ-Tcl1) and human (CLL) leukemia cells. Furthermore, PI3Kδi imparted significant therapeutic responses in Eμ-Tcl1-bearing animals and enhanced anti-CD20 monoclonal antibody therapy. Responses correlated with upregulation of the pro-apoptotic BH3-only protein Bim. Accordingly, Bim−/− Eμ-Tcl1 Tg leukemias demonstrated resistance to PI3Kδi-induced apoptosis were refractory to PI3Kδi in vivo and failed to display combination efficacy with anti-CD20 monoclonal antibody therapy. Therefore, Bim-dependent apoptosis represents a key in vivo therapeutic mechanism for PI3Kδi, both alone and in combination therapy regimes.

Green tea polyphenol “epigallocatechin-3-gallate”, differentially induces apoptosis in CLL B-and T-Cells but not in healthy B-and T-Cells in a dose dependant manner

B-cell chronic lymphocytic leukaemia (CLL) is characterized by an accumulation of CD5-positive monoclonal B-cells due in large part to a failure of apoptosis. The ability to study CLL B-cells in vitro has always been a challenge and hampered by the low viability of the CLL B-cells in cell culture systems. In this study, we present a multicellular cell culture system to maintain CLL B-cells viable in culture for 60h in the presence of a stromal cell feeder layer in combination with a whole white blood cell preparation. Using this optimized system, we tested and showed that the addition of epigallocatechin-3-gallate (EGCG) at concentrations ranging from 25 to 100μg/ml induced apoptosis in CLL B-cells whilst not affecting healthy control B-cells. Moreover, the results showed that in contrast to healthy controls, T-cells from CLL patients underwent apoptosis in the presence of EGCG. This study demonstrated that the combination of a cell feeder layer with a whole white blood cell preparation maintained B-cell viability in vitro over an extended period of time. In addition, the study showed that EGCG differentially induces apoptosis in CLL B-and T-Cells but not in healthy B-and T-Cells in a dose dependent manner.

Inhibition of JAK3 and PKC via Immunosuppressive Drugs Tofacitinib and Sotrastaurin Inhibits Proliferation of Human B Lymphocytes In Vitro

BackgroundAntibody-mediated response in solid organ transplantation is critical for graft dysfunction and loss. The use of immunosuppressive agents partially inhibits the B-lymphocyte response leading to a risk of acute and chronic antibody-mediated rejection. This study evaluated the impact of JAK3 and PKC inhibitors tofacitinib (Tofa) and sotrastaurin (STN), respectively, on B-cell proliferation, apoptosis, and activation in vitro. MethodsHuman B cells isolated from peripheral blood of healthy volunteers were cocultured with CD40 ligand–transfected fibroblasts as feeder cells in the presence of interleukin (IL) 2, IL-10, and IL-21. The cocultures were treated with immunosuppressants Tofa, STN, and rapamycin (as a control), to analyze the proliferation and apoptosis of B cells by means of Cyquant and flow cytometry, respectively. CD27 and IgG staining were applied to evaluate whether treatments modified the activation of B cells. ResultsTofa and STN were able to inhibit B-cell proliferation to the same extent as rapamycin, without inducing cell apoptosis. After 6 days in coculture with feeder cells, all B cells showed CD27 memory B-cell phenotype. None of the immunosuppressive treatments modified the proportion between class-switched and non–class-switched memory B cells observed in nontreated cultures. The high predominance of CD27+CD24+ phenotype was not modified by any immunosuppressive treatment. ConclusionsOur results show that Tofa and STN can suppress B-cell antibody responses to an extent similar to rapamycin, in vitro; therefore these compounds may be a useful therapy against antibody-mediated rejection in transplantation.

CD95-mediated apoptosis in Burkitt's lymphoma B-cells is associated with Pim-1 down-regulation

B-cells of the high-grade non-Hodgkin lymphoma Burkitt's lymphoma (BL) overexpress survival oncoproteins, including the proviral integration site for Moloney murine leukaemia virus kinase (Pim)-1, and become apoptosis resistant. Activated death receptor CD95 after ligation with anti-CD95 monoclonal antibody (mAb) resulted in the regression of BL via induction of apoptosis, suggesting a decrease of survival protein expression. Here, CD95-mediated apoptotic pathways in BL B-cell lines (Raji and Daudi) following treatment with anti-CD95 mAb was investigated with the cause-and-effects on pim-1 gene expression, in comparison with leukemic cell line (K562) used as CD95-negative cells. Immunohistochemical staining for CD95 and Pim-1 was performed, and the effects of anti-CD95 mAb on apoptotic signalling using western blotting, on caspase activity and cell survival of BL B-cell and leukemic cell lines were determined. We showed that Raji cells expressed more CD95 receptors than Daudi cells. Half of each population underwent apoptosis accompanied by decreased cell viability after anti-CD95 mAb treatment. Distinct extrinsic and intrinsic CD95-mediated apoptotic pathways in Raji and Daudi cells were revealed by high caspase activity and mitochondrial outer membrane permeabilization, respectively. We observed decreased Pim-1 transcript and protein expression levels with increased heat-shock protein (Hsp)70 and decreased Hsp90 expression in anti-CD95 mAb-treated cells. Throughout the study, K562 cells did not undergo apoptosis upon anti-CD95 mAb treatment. Pim-1 knockdown following to stable transfection with plasmid vectors induced apoptosis and decreased viability of BL and K562 cells. Therefore, CD95-mediated apoptosis induces Pim-1 down-regulation in BL B-cells, but Pim-1 down-regulation cannot fully eradicate BL and leukaemia.

Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells.

Crosslinking of B-cell receptor (BCR) sets off an apoptosis programme, but the underlying pathways remain obscure. Here we decipher the molecular mechanisms bridging B-cell activation and apoptosis mediated by post-translational modification (PTM). We find that O-GlcNAcase inhibition enhances B-cell activation and apoptosis induced by BCR crosslinking. This proteome-scale analysis of the functional interplay between protein O-GlcNAcylation and phosphorylation in stimulated mouse primary B cells identifies 313 O-GlcNAcylation-dependent phosphosites on 224 phosphoproteins. Among these phosphoproteins, temporal regulation of the O-GlcNAcylation and phosphorylation of lymphocyte-specific protein-1 (Lsp1) is a key switch that triggers apoptosis in activated B cells. O-GlcNAcylation at S209 of Lsp1 is a prerequisite for the recruitment of its kinase, PKC-β1, to induce S243 phosphorylation, leading to ERK activation and downregulation of BCL-2 and BCL-xL. Thus, we demonstrate the critical PTM interplay of Lsp1 that transmits signals for initiating apoptosis after BCR ligation.