B-cell Receptor Ligation Research Articles

BCR ligation selectively inhibits IgE class switch recombination.

Mechanisms that restrict class switch recombination (CSR) to IgE limit the subsequent production of IgE antibodies and therefore the development of allergic disease. Mice with impaired B cell receptor (BCR) signaling have significantly increased IgE responses, consistent with a role for BCR signaling in IgE regulation. While prior work focused on BCR signaling in IgE-expressing cells to explain these findings, it has been reported that BCR signaling can reduce CSR. Therefore, we investigated the possibility that IgE CSR might be particularly sensitive to inhibition by BCR signaling in unswitched B cells. We found that immunization of mice with high-affinity antigen resulted in reduced representation of IgE-expressing cells among germinal center B cells and plasma cells relative to a low-affinity antigen. Mechanistic experiments with cultured mouse B cells demonstrated that BCR ligands selectively inhibited IgE CSR in a dose-, affinity-, and avidity-dependent manner. Signaling via Syk was required for the inhibition of IgE CSR following BCR stimulation, whereas inhibition of the PI3K subunit p110δ increased IgE CSR independently of BCR ligation. The inhibition of IgE CSR by BCR ligands synergized with IL-21 or TGFβ1. BCR ligation also inhibited CSR to IgE in human tonsillar B cells, and this inhibition was also synergistic with IL-21. These findings establish that IgE CSR is uniquely susceptible to inhibition by BCR signaling in mouse and human B cells, with important implications for the regulation and pathogenesis of allergic disease.

A mechanism for the elimination of IgE plasma cells

Abstract The proper regulation of IgE production safeguards against allergic disease, highlighting the importance of mechanisms that restrict IgE plasma cell (PC) survival. IgE PCs have unusually high surface B cell receptor (BCR) expression, yet the functional consequences of ligating this receptor are unknown. Here, we found that BCR ligation induced BCR signaling in IgE PCs followed by their elimination. In cell culture, exposure of IgE PCs to cognate antigen or anti-BCR antibodies induced apoptosis. IgE PC depletion correlated with the affinity, avidity, amount, and duration of antigen exposure and required the BCR signalosome components Syk, BLNK, and PLCγ2. In mice with a PC-specific impairment of BCR signaling, the abundance of IgE PCs was selectively increased. Conversely, BCR ligation by injection of cognate antigen or anti-IgE depleted IgE PCs. These findings establish a mechanism for the elimination of IgE PCs through BCR ligation. This has important implications for allergen tolerance and immunotherapy as well as anti-IgE monoclonal antibody treatments. This research was supported by the National Institute of Allergy and Infections Diseases of the National Institutes of Health under Award Number R01AI130470; the Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation; the Sandler Asthma Basic Research Center; and the Cardiovascular Research Institute at the University of California, San Francisco. C.D.C.A. was a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts. A.K.W-V was supported by a Doctoral Foreign Study Award from the Canadian Institute of Health Research, funding reference number DFD-170769. D.M.T. and M.J.R. were funded by National Health and Medical Research Council (NHMRC) Australia by an Investigator Award (APP1175411) and Ideas Grant (APP1185294), respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

B cell receptor ligation induces IgE plasma cell elimination.

The proper regulation of IgE production safeguards against allergic disease, highlighting the importance of mechanisms that restrict IgE plasma cell (PC) survival. IgE PCs have unusually high surface B cell receptor (BCR) expression, yet the functional consequences of ligating this receptor are unknown. Here, we found that BCR ligation induced BCR signaling in IgE PCs followed by their elimination. In cell culture, exposure of IgE PCs to cognate antigen or anti-BCR antibodies induced apoptosis. IgE PC depletion correlated with the affinity, avidity, amount, and duration of antigen exposure and required the BCR signalosome components Syk, BLNK, and PLCγ2. In mice with a PC-specific impairment of BCR signaling, the abundance of IgE PCs was selectively increased. Conversely, BCR ligation by injection of cognate antigen or anti-IgE depleted IgE PCs. These findings establish a mechanism for the elimination of IgE PCs through BCR ligation. This has important implications for allergen tolerance and immunotherapy as well as anti-IgE monoclonal antibody treatments.

Evidence of a Synergistic Cross-Talk between the B Cell Receptor (BCR) and Nicotinamide Phosphoribosyl Transferase (NAMPT) in Richter's Syndrome Patient-Derived Xenograft Models: Therapeutic Implications

Background: A rare complication of Chronic Lymphocytic Leukemia (CLL), Richter's Syndrome (RS) represents the transformation of a pre-existing CLL into a Diffuse Large B-cell Lymphoma (DLBCL), generally associated with poor prognosis. Current therapeutic approaches are limited and do not significantly reduce disease progression. For these reasons there is intense investigation to identify potentially druggable molecular circuits, opening the way to innovative combination therapies.Among the several oncogenic signalling pathways that may contribute to disease progression, the B Cell Receptor (BCR) is a main driver and an actionable target. We previously showed that BCR ligation in CLL cells increases expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage NAD pathway starting from nicotinamide, a finding in line with the notion of oncogenic-driven metabolic reprogramming. In this context, increased NAMPT expression leading to heightened NAD+ levels could sustain proliferation through the modulation of the activity of several intracellular enzymes, including sirtuins (NAD-dependent deacetylases).Aim: This work explores the connections between BCR signalling and the NAMPT/NAD/sirtuin axis in RS cells, asking the question of whether BCR and NAMPT can be simultaneously targeted.Methods: RS-PDX cells were freshly purified from tumor masses grown in mice and immediately used for short ex-vivo experiments. For in vivo experiments, NSG mice were injected subcutaneously with RS-PDX cells, which were left to engraft until a palpable mass was evident. Mice were then randomized to receive duvelisib, OT82 or a combination of the two for two consecutive weeks. Control mice were similarly treated with vehicle only. Mice were under a niacin-free diet.Results: By using our 4 RS patient-derived xenografts (PDX) models, we invariably observed high levels of NAMPT expression. High levels of NAMPT expression were also observed in 15 primary RS lymph node biopsies analyzed by RNA sequencing. BCR engagement through αIgM polyclonal antibodies significantly up-regulated NAMPT expression, as determined by qRT-PCR and protein analysis, with a concomitant increase in intracellular NAD+ levels. We then asked whether RS cells are sensitive to NAMPT inhibition, alone or in combination with drugs that target the BCR pathway. As most RS patients would likely have been treated during the preceding CLL phase with a BTK inhibitor, possibly developing resistance, we turned to PI3K inhibitors, which are less commonly used for CLL therapy. As NAMPT inhibitors (NAMPTi) we used both FK866 and OT-82, which are validated small molecules. Results indicated that the combination of the dual PI3K-δ/γ inhibitor duvelisib, with either FK866 or OT-82 induces dramatic apoptosis in all 4 models tested, as confirmed by annexinV/PI staining, by caspase 3 activation and by a significant drop in ATP and NAD+ levels. Importantly, two RS-PDX models (RS9737 and RS1316) were fully resistant to NAMPTi used alone, likely due to high levels of nicotinate phosphoribosyltransferase (NAPRT), which is the rate-limiting enzyme in the NAD salvage pathway that starts from nicotinic acid. However, addition of duvelisib, which was mildly effective when used alone, was followed by marked apoptosis even in these two models.Molecular dissection of the pathway showed that the combination of duvelisib and NAMPTi was followed by complete inhibition of the PI3K pathway, which was only partially blocked by duvelisib alone, even at high doses. The connection between NAMPT and PI3K is represented by cytoplasmic sirtuins, particularly SIRT2, which activate AKT through de-acetylation. Immunoprecipitation and two-dimensional gel electrophoresis showed that in the presence of NAMPTi, the amount of acetylated, i.e., inactive, AKT increased considerably. Consistently, treatment of RS-PDX mice with a combination of duvelisib and OT-82 was followed by significantly higher responses and longer animal survival.Conclusions: These results highlight a crosstalk between BCR signalling and NAMPT/sirtuin axis in RS models, showing the increased efficacy of the dual targeting (i.e., PI3K-δ/γ and NAMPT), and supporting this novel and promising therapeutic strategy for the treatment of RS patients. DisclosuresDeaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

MiR-29 modulates CD40 signaling in chronic lymphocytic leukemia by targeting TRAF4: an axis affected by BCR inhibitors

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

Lipid Rafts Interaction of the ARID3A Transcription Factor with EZRIN and G-Actin Regulates B-Cell Receptor Signaling.

Several diseases originate via dysregulation of the actin cytoskeleton. The ARID3A/Bright transcription factor has also been implicated in malignancies, primarily those derived from hematopoietic lineages. Previously, we demonstrated that ARID3A shuttles between the nucleus and the plasma membrane, where it localizes within lipid rafts. There it interacts with components of the B-cell receptor (BCR) to reduce its ability to transmit downstream signaling. We demonstrate here that a direct component of ARID3A-regulated BCR signal strength is cortical actin. ARID3A interacts with actin exclusively within lipid rafts via the actin-binding protein EZRIN, which confines unstimulated BCRs within lipid rafts. BCR ligation discharges the ARID3A–EZRIN complex from lipid rafts, allowing the BCR to initiate downstream signaling events. The ARID3A–EZRIN interaction occurs almost exclusively within unpolymerized G-actin, where EZRIN interacts with the multifunctional ARID3A REKLES domain. These observations provide a mechanism by which a transcription factor directly regulates BCR signaling via linkage to the actin cytoskeleton with consequences for B-cell-related neoplasia.

SLAMF receptors negatively regulate B cell receptor signaling in chronic lymphocytic leukemia via recruitment of prohibitin-2

We identified a subset of Chronic Lymphocytic Leukemia (CLL) patients with high Signaling Lymphocytic Activation Molecule Family (SLAMF) receptor-related signaling that showed an indolent clinical course. Since SLAMF receptors play a role in NK cell biology, we reasoned that these receptors may impact NK cell-mediated CLL immunity. Indeed, our experiments showed significantly decreased degranulation capacity of primary NK cells from CLL patients expressing low levels of SLAMF1 and SLAMF7. Since the SLAMFlow signature was strongly associated with an unmutated CLL immunoglobulin heavy chain (IGHV) status in large datasets, we investigated the impact of SLAMF1 and SLAMF7 on the B cell receptor (BCR) signaling axis. Overexpression of SLAMF1 or SLAMF7 in IGHV mutated CLL cell models resulted in reduced proliferation and impaired responses to BCR ligation, whereas the knockout of both receptors showed opposing effects and increased sensitivity toward inhibition of components of the BCR pathway. Detailed molecular analyzes showed that SLAMF1 and SLAMF7 receptors mediate their BCR pathway antagonistic effects via recruitment of prohibitin-2 (PHB2) thereby impairing its role in signal transduction downstream the IGHV-mutant IgM-BCR. Together, our data indicate that SLAMF receptors are important modulators of the BCR signaling axis and may improve immune control in CLL by interference with NK cells.

Bidirectional linkage between the B-cell receptor and NOTCH1 in chronic lymphocytic leukemia and in Richter’s syndrome: therapeutic implications

NOTCH1 mutations in chronic lymphocytic leukemia (CLL) lead to accumulation of NOTCH1 intracellular domain (NICD) and prolong signaling. These mutations associate with a more aggressive disease compared to wild-type (WT) CLL. In this work we demonstrate a bidirectional functional relationship between NOTCH1 and the B cell receptor (BCR) pathways. By using highly homogeneous cohorts of primary CLL cells, activation of NOTCH1 is shown to increase expression of surface IgM, as well as LYN, BTK, and BLNK, ultimately enhancing BCR signaling responses, including global mRNA translation. Upon BCR cross-linking, NOTCH1 itself is actively translated and increased on cell surface. Furthermore, BCR ligation induces calcium mobilization that can facilitate ligand-independent NOTCH1 activation. These data suggest that the two pathways are functionally linked, providing a rationale for dual inhibition strategies. Consistently, addition of the γ-secretase inhibitor DAPT to ibrutinib significantly potentiates its effects, both in vitro and in a short-term patient-derived xenograft model. While this observation may find limited applications in the CLL field, it is more relevant for Richter's Syndrome (RS) management, where very few successful therapeutic options exist. Treatment of RS-patient-derived xenografts (RS-PDX) with the combination of ibrutinib and DAPT decreases disease burden and increases overall survival.

NOTCH1 Stabilization By PEST Mutations Enhances IgM-Mediated Activity in Chronic Lymphocytic Leukemia

BACKGROUND. Mutations in NOTCH1 PEST domain (NOTCH1-M) are present in ~10% of Chronic Lymphocytic Leukemia (CLL) patients, result in accumulation of more stable NOTCH1 protein, and associate with poorer prognosis. NOTCH1-M are enriched in unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) CLL, which show high surface IgM (sIgM) expression and signaling capacity. mRNA translation is a prominent response to B cell receptor (BCR) engagement, increased in U-CLL, and for which therapeutic inhibitors are under active development. In CLL, c-MYC is an essential mediator of BCR-driven translation and direct target of NOTCH1, suggesting the impact of NOTCH1 on anti-IgM-mediated cell growth via MYC.AIMS AND METHODS. Our aim was to investigate the functional role of NOTCH1-M on anti-IgM-mediated signaling, compared to wild-type (WT) NOTCH1. The impact on global mRNA translation was studied using a flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay and polysome fractionation assays. The effects of stabilized vs WT NOTCH1 were measured after 24-hour cultures of CLL cells, when data demonstrate differences in the expression of the two forms. Two cohorts of U-CLLs were compared: i) a subset of samples carrying NOTCH1-M [variant allele frequency (VAF) ≥30%, n=21] and ii) a cohort of samples with WT NOTCH1 (VAF<1%, n=23). In both subsets no additional cytogenetic lesions other than 13q deletion were present.RESULTS. sIgM levels and signaling capacity (measured by anti-IgM mediated iCa2+ mobilization) were higher in NOTCH1-M than in -WT samples, consistent with previous observations (1). Conceivably, anti-IgM-mediated phosphorylation of PLCg2 and ERK1/2 was stronger in M than in WT CLLs. In keeping with these results, expression of downstream targets as MYC and CCL3 was also induced at higher levels in M samples. Interestingly, inhibition of NOTCH1 with g-secretase inhibitor (DAPT) significantly decreased BCR target genes induction in M cells, reducing the differences with WT samples, and further enhanced the effects of ibrutinib when used in combination. In order to investigate the impact of NOTCH1 on IgM-mediated CLL cell growth, anti-IgM-induced global mRNA translation was compared in the two cohorts. Consistent with the higher MYC mRNA and protein levels, anti-IgM led to higher global mRNA translation in NOTCH1-M than in -WT cells. DAPT inhibited it in both CLL subsets, while ibrutinib led to complete inhibition of mRNA translation only in the -WT subset, suggesting a major contribution of NOTCH1 to the process. Consistently, the combination of DAPT+ibrutinib abrogated the difference between M and WT CLL cells.Importantly, MYC (but not translation initiation factors eIF4G, eIF4A or eIF3b) was already induced at 6 hours following anti-IgM stimulation and was maintained at high levels at 24 hours, while up-regulation of eIF4G, eIF4A and eIF3b was evident only at 24 hours, supporting the hypothesis of a direct MYC-dependent regulation of the translation machinery (2).NOTCH1 itself was post-transcriptionally regulated upon BCR ligation, as we observed increased NOTCH1 mRNA in polysome-enriched actively translated fractions and increased protein levels on the surface of anti-IgM stimulated cells, specifically inhibited by ibrutinib. Consequently, NOTCH1 pathway was significantly more activated upon anti-IgM stimulation in M than WT cells, as determined by qPCR of NOTCH1 target genes. Both Ibrutinib and DAPT significantly prevented NOTCH1 activation upon BCR triggering, with the drug combination being the most effective treatment. Moreover, in line with data showing NOTCH1-dependent regulation of a B cell gene signature, expression of BTK, LYN and BLNK was significantly increased in anti-IgM activated NOTCH1-M samples, an effect prevented by DAPT.CONCLUSIONS. These data indicate that NOTCH1 stabilization associates with stronger IgM signaling capacity and suggest an interplay between BCR and NOTCH1 pathway, with the former promoting NOTCH1 expression and activation. The evidence that NOTCH1 pathway inhibition reverts this difference suggests a direct effect of NOTCH1 on IgM signaling. In this scenario, stabilizing NOTCH1 mutations may enhance BCR signaling by boosting translation through MYC induction and by directly regulating expression of BCR cascade elements.NOTES. SD and FF share senior authorshipD'Avola, Blood 2016Ruggero, Cancer Res 2009 DisclosuresCoscia:Abbvie, Gilead, Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen, Karyopharm: Research Funding. Gaidano:Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Allan:Genentech: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy; Verastem: Membership on an entity's Board of Directors or advisory committees. Furman:Gilead: Consultancy; AbbVie: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Incyte: Consultancy, Other: DSMB; Loxo Oncology: Consultancy; TG Therapeutics: Consultancy; Sunesis: Consultancy; Acerta: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy. Packham:Aquinox: Research Funding. Deaglio:iTeos therapeutics: Research Funding; VelosBio inc: Research Funding; Verastem: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.

DEC1/STRA13 is a key negative regulator of activation-induced proliferation of human B cells highly expressed in anergic cells

The transcription factor DEC1/STRA13 (also known as BHLHE40 and SHARP2) is involved in a number of processes including inhibition of cell proliferation and delay of cell cycle, and is a negative regulator of B cell activation and development in mice. We show here that, unlike in mice, DEC1/STRA13 expression is induced in human naïve and memory resting B cells by activation through the B-cell receptor (BCR) or Toll-like receptor 9 (TLR9). siRNA silencing of DEC1/STRA13 increases the capacity of activated B cells to perform a high number of divisions after TLR9 ligation. This identifies DEC1/STRA13 as a critical negative regulator of clonal expansion of activated human B cells. We also show that DEC1/STRA13 is upregulated in human anergic CD21low B cells clonally expanded in patients with HCV-associated mixed cryoglobulinemia, which fail to proliferate in response to BCR or TLR9 ligation. siRNA knockdown of DEC1/STRA13, however, fails to restore responsiveness to stimuli in these cells, although it might improve the proliferative capacity in a subset of anergic cells with less pronounced proliferative defect.

B Cell Receptor and CD40 Signaling Are Rewired for Synergistic Induction of the c-Myc Transcription Factor in Germinal Center B Cells

Positive selection of germinal center (GC) B cells is driven by B cell receptor (BCR) affinity and requires help from follicular T helper cells. The transcription factors c-Myc and Foxo1 are critical for GC B cell selection and survival. However, how different affinity-related signaling events control these transcription factors in a manner that links to selection is unknown. Here we showed that GC B cells reprogram CD40 and BCR signaling to transduce via NF-κB and Foxo1, respectively, whereas naive B cells propagate both signals downstream of either receptor. Although either BCR or CD40 ligation induced c-Myc in naive B cells, both signals were required to highly induce c-Myc, a critical mediator of GC B cell survival and cell cycle reentry. Thus, GC B cells rewire their signaling to enhance selection stringency via a requirement for both antigen receptor- and Tcell-mediated signals to induce mediators of positive selection.

Reversion of anergy signatures in clonal CD21low B cells of mixed cryoglobulinemia after clearance of HCV viremia

AbstractHepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of IgM+CD27+ B cells. These cells display both the features of anergy induced by continual engagement of the B-cell receptor (BCR), such as high expression of phosphorylated extracellular signal–regulated kinase (pERK) and reduced lifespan, and of virus-specific exhaustion, such as CD21low phenotype and a defective response to ligation of BCR and Toll-like receptor 9 (TLR9). MC usually regresses after eradication of HCV with interferon, whose immunomodulatory activity might contribute to this effect. We investigated the phenotypic and functional changes in clonal B cells of MC patients with sustained virologic responses to direct-acting antivirals (DAAs), which lack immunomodulatory properties. We found that high pERK expression and accelerated apoptosis revert within 4 weeks after beginning therapy, whereas clonal B cells unresponsive to TLR9 stimulation persist for at least 24 weeks, although they may partially rescue normal CD21 expression. Thus, similar to mouse models, features of anergy in MC B cells rapidly revert after disengagement from HCV, whereas virus-specific exhaustion imparts a durable inhibitory imprint on cell function. Treatment of HCV+ MC with DAAs provides a valuable tool for untangling the molecular mechanisms of anergy and exhaustion in human B cells.

Exosomes from B lymphocytes are a major source of Fas ligand

Abstract Exosomes are nanovesicles released by a variety of cell types, including antigen-presenting cells. The ability of exosomes to induce immunity has prompted great interest in their clinical use. Murine studies have also indicated that circulating FasL+MHCII+ exosomes can be tolerogenic in allergic and delayed-type hypersensitivity responses. B cells are capable of expressing FasL and are a major in vivo source of exosomes, but their role in producing tolerogenic exosomes is incompletely explored. In the current study, we evaluated B cells as a source of FasL+ exosomes in vivo and signals that induced B cells to release FasL+ exosomes in vitro. In B-cell deficient mice, a major loss in total splenic exosomes and exosome-associated FasL was observed, suggesting B cells are the major source of FasL+ exosomes in vivo. After sorting B cells into CD107a+, CD5+ or CD9+ cells, all subsets showed some intracellular storage of FasL, yet FasL was predominately found in the CD9+ subset. Searching for signals that induced FasL+ exosomes in vitro, we stimulated B cell signaling through Toll-like receptors (TLRs), the B-cell receptor (BCR), and/or CD40. In response to BCR ligation, B cells produced 20% more FasL compared to medium controls, which increased to 50% in the presence of any TLR ligand. Interestingly, B cells increased their intracellular FasL storage, but failed to increase their FasL+ exosome release when stimulated through CD40 alone. TLR-4, TLR-7(/8) or TLR-9 ligation with CD40 activation induced the FasL+ exosome release, which was further enhanced by BCR ligation. These data suggest that signals known to induce the differentiation of IL-10-producing B cells, also enhance FasL+ exosome production as another potential suppressive mechanism.

Myc enhances B-cell receptor signaling in precancerous B cells and confers resistance to Btk inhibition.

Dysregulation of the oncogenic transcription factor MYC induces B cell transformation and is a driver for B cell non-Hodgkin lymphoma (B-NHL). MYC overexpression in B-NHL is associated with more aggressive phenotypes and poor prognosis. Although genomic studies suggest a link between MYC overexpression and B cell receptor (BCR) signaling molecules in B-NHL, signaling pathways essential to Myc-mediated B-cell transformation have not been fully elucidated. We utilized intracellular phospho-flow cytometry to investigate the relationship between Myc and BCR signaling in pre-malignant B cells. Utilizing the Eμ-myc mouse model, where Myc is overexpressed specifically in B cells, both basal and stimulated BCR signaling were increased in precancerous B lymphocytes from Eμ-myc mice compared to wild-type littermates. B cells overexpressing Myc displayed constitutively higher levels of activated CD79α, Btk, Plcγ2, and Erk1/2. Notably, Myc overexpressing B cells maintained elevated BCR signaling despite treatment with ibrutinib, a Bruton’s tyrosine kinase inhibitor. Furthermore, PI3K/Akt pathway signaling was also increased in Eμ-myc B cells, and this increase was partially suppressed with ibrutinib. Additionally, experiments with Btk-null B cells revealed off-target effects of ibrutinib on BCR signaling. Our data show that in pre-malignant B cells, Myc overexpression is sufficient to activate BCR and PI3K/Akt signaling pathways and further enhances signaling following BCR ligation. Therefore, our results indicate precancerous B cells have already acquired enhanced survival and growth capabilities prior to transformation, and that elevated MYC levels confer resistance to pharmacologic inhibitors of BCR signaling, which has significant implications for B-NHL treatment.

Uncoupling of BCR Internalization and Signaling in Treatment Naive and Btki-Treated Patients with Chronic Lymphocytic Leukemia

Ligation of the B-cell receptor (BCR) results in activation of intracellular signaling as well as internalization and processing of ligand/receptor complexes. BCR responsiveness has been shown to vary markedly between patients with chronic lymphocytic leukaemia (CLL) and is linked to prognosis. Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, relatively little is known about the capacity of CLL B-cells to internalize ligands that bind to the BCR.In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR following stimulation. First, we assessed to what extent this varies between various prognostic subgroups. Second, given that BCR signaling is thought to be more pronounced within lymphoid tissue, we investigated whether internalization varies between different anatomic sites of the same individual. Finally, we examined the effect of agents that inhibit BCR function by comparing BCR expression and internalization in a cohort of patient before and during therapy with Bruton's tyrosine kinase inhibitors (BTKi).BCR internalization was assessed in two ways. First, we used a pH sensitive dye linked to agonistic anti-IgM (pHrodo-αIgM) to detect the uptake and retention of ligand/receptor complexes in acidified endosomes. Second, BCR internalization was assessed directly by measuring the rate of disappearance of surface IgM following ligation by agonistic anti-IgM. An increase in the percentage of cells showing pHrodo fluorescence above control was detected in all CLL cases studied (mean percentage pHrodo-αIgM uptake = 30.2±2.5%, p<0.0001, n=40) although there was considerable inter-patient variation. We observed a significant correlation between pHrodo-αIgM uptake and high surface IgM (sIgM) expression (r2=0.529, p=0.0001, n=40), and an association with markers of adverse prognosis including, CD38 positivity (p=0.001, n=40), and the capacity to mobilize calcium ions following BCR ligation (p=0.001, n=26). Cases of CLL with unmutated IGHV genes showed higher uptake of pHrodo-αIgM than mutated cases however this did not reach statistical significance (p=0.053, n=40).Similarly, when BCR function within individual CLL patients was examined, we also found that pHrodo-αIgM uptake varied substantially and was maximal in lymph node (LN) derived CLL cells (p=0.03, n=6) and those in the peripheral blood (PB) that express the highest levels of CD5 (p=0.0001, n=26), a marker that is upregulated following BCR activation. In addition, we found that LN CLL cells expressed higher levels of sIgM than those derived from the PB (p=0.03, n=6). This was a surprising finding, as BCR stimulation is thought to occur within LNs, which might be expected to result in down regulated BCR expression.When the level of BCR internalization and accumulation in the endosomes was adjusted for the number of sIgM molecules, we found that BCR internalization and retention was actually more efficient in anergic cases of CLL (defined by lack of ability to mobilize Ca2+ following stimulation with anti-IgM; p=0.0002, n=26). This observation was supported by direct measurement of the rate of BCR endocytosis, which showed a more rapid internalization in anergic B-cells compared to signaling competent and normal B-cells. Similar findings have previously been reported in a murine model of B-cell anergy.Finally, data from BTKi treated CLL patients showed that, 12 months after commencing treatment, CLL B-cells exhibit lower levels of sIgM expression (p=0.02, n=7) and have more efficient BCR internalization than at the outset (p=0.05, n=7). Since low sIgM expression and more efficient BCR internalization is a feature of B-cell anergy, these results suggest that therapy with BTK inhibitors selectively depletes B-cells that are capable of signaling through the BCR and enriches for those displaying features of anergy.Overall our data show that BCR internalization is uncoupled from intracellular signaling in CLL and is most efficient in cells that demonstrate poor downstream BCR signaling or show features of recent BCR stimulation. These observations are similar to those previously reported in anergic B-cells and provide further evidence for ongoing BCR activation and anergy in CLL. DisclosuresPatten:Gilead: Research Funding. Devereux:Gilead: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Consultancy, Other: Travel, Accommodations, Expenses ; GSK: Consultancy.

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

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.

Dynamics of microvesicle generation in B-cell chronic lymphocytic leukemia: implication in disease progression.

Previously, we reported that B-cell chronic lymphocytic leukemia (CLL) patients contained elevated levels of microvesicles (MVs). However, given the quiescent nature of CLL B-cells and the relative indolence of the disease, the dynamics of MV generation and their unique phenotypes are not clearly defined. In this study, we find that CLL B-cells generate MVs spontaneously and can be further induced by B-cell receptor-ligation. Most interestingly, CLL B-cells predominantly generate CD52+ MVs, but not CD19+ MVs in vitro, suggesting preferential usage of CD52 into leukemic-MVs and that the CLL plasma MV phenotypes corroborate well with the in vitro findings. Importantly, we detected increased accumulation of CD52+ MVs in previously untreated CLL patients with progressive disease. Finally, sequential studies on MVs in pre- and post-therapy CLL patients demonstrate that while the plasma CD52+ MV levels drop significantly after therapy in most and remain at low levels in some patients, a trend of increased accumulation of CD52+ MVs was detected in majority of post-therapy CLL patients (25 of 33). In total this study emphasizes that dynamic accumulation of CD52+ MVs in plasma can be used to study CLL progression and may be a useful biomarker for patients as they progress and require therapy.

Mimicking the tumour microenvironment of chronic lymphocytic leukaemia in vitro critically depends on the type of B-cell receptor stimulation.

Background:The B-cell receptor (BCR) has a key role in the cross-talk between chronic lymphocytic leukaemia (CLL) cells and the tissue microenvironment, which favours disease progression by promoting proliferation and drug resistance. In vitro studies on downstream signalling and functional effects of CLL BCR ligation often report contradictory results, in part owing to the lack of a standardised stimulation protocol. Our aim was to define a biologically relevant and robust in vitro stimulation method with regard to cellular phenotypic and transcriptional responses.Methods:We evaluated mRNA (FOS, MYC, LPL) and protein (CD54, CD19, CD62L, CD184) expression of genes modulated by BCR triggering in immunoglobulin heavy-chain variable region genes (IGHV)-mutated and -unmutated CLL cells, after stimulation using soluble or immobilised anti-IgM antibodies from different suppliers.Results:The effect of BCR stimulation on gene and protein expression was comparable in all CLL patients, irrespective of IGHV mutation status. However, immobilised anti-IgM stimulation elicited clear and robust changes in gene and protein expression, whereas the response to soluble anti-IgM was far less obvious.Conclusions:These data indicate that the method of BCR stimulation is of major importance regarding responsiveness of CLL cells in the context of the tumour microenvironment, whereas genetic differences in the BCR pathway are less critical.

Abstract B85: EZH2 plays a critical role in B-cell maturation and in non-Hodgkin's lymphoma: Interplay between EZH2 function and B-cell activation

Abstract The EZH2 inhibitor tazemetostat (EPZ-6438) is emerging as a promising therapeutic agent for the treatment of non-Hodgkin's Lymphoma (NHL). A significant body of work has now demonstrated in vitro and in vivo effects of EZH2 inhibition in preclinical models of lymphoma, in addition to objective clinical responses in early human trials. While EZH2 gain-of-function mutations clearly contribute to lymphomagenesis, patients with lymphomas harboring wild-type EZH2 also show responses to tazemetostat. This suggests a broad role of EZH2 in B-cell oncogenesis. Several recent mouse model studies have demonstrated the importance of wild-type EZH2 catalytic activity in the formation of germinal centers in non-diseased lymph nodes, suggesting a central role for EZH2 in B-lymphocyte maturation. These findings indicate that the importance of EZH2 to B-cell lymphoma likely lies in its ability to regulate B-cell differentiation. To understand the relationship between B-cell maturation and sensitivity to EZH2 inhibition, we evaluated changes in maturation markers and cellular proliferation following treatment of diffuse large B-cell lymphoma (DLBCL) cell lines with tazemetostat in combination with modulators of B-cell activation. Consistent with the importance of EZH2 in the regulation of B-cell differentiation, we observed increased expression of B-cell maturation markers in DLBCL cell lines treated with single agent tazemetostat in vitro. Furthermore, we demonstrate that tazemetostat pre-treatment of subsets of DLBCL cells lines (both EZH2 mutant and wild-type) can sensitize cells to inhibitors of B-cell activation pathways, which include glucocorticoids and BTK, MAPK and PI3K pathway inhibitors. Moreover, the anti-proliferative activity of single agent tazemetostat can be diminished or delayed by co-treatment with biological stimulators of B-cell activation including B-cell receptor ligation, CD40L, LPS and BAFF. Importantly, B-cell receptor ligation and co-stimulation agents have little proliferative effects on DLBCL cell lines on their own, suggesting that the protective function of these agents is directly related to the effects of EZH2 inhibition and not a generic stimulation of proliferation. Our findings suggest that EZH2 inhibition initiates a differentiation program that enables lymphoma cells to proceed through the normal processes of B-cell selection, growth regulation and maturation. Citation Format: Danielle Johnston, Dorothy Brach, Christopher Plescia, Alison Drew, Trupti Lingaraj, Natalie Warholic, Jesse J. Smith, Robert A. Copeland, Heike Keilhack, Elayne Penebre, Sarah K. Knutson, Scott Ribich, Michael J. Thomenius, Alejandra Raimondi. EZH2 plays a critical role in B-cell maturation and in non-Hodgkin's lymphoma: Interplay between EZH2 function and B-cell activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B85.