B-cell Antigen Receptor Research Articles

Cell signaling and the aging of B cells

The uniqueness of each B cell lies in the structural diversity of the B-cell antigen receptor allowing the virtually limitless recognition of antigens, a necessity to protect individuals against a range of challenges. B-cell development and response to stimulation are exquisitely regulated by a group of cell surface receptors modulating various signaling cascades and their associated genetic programs. The effects of these signaling pathways in optimal antibody-mediated immunity or the aberrant promotion of immune pathologies have been intensely researched in the past in young individuals. In contrast, we are only beginning to understand the contribution of these pathways to the changes in B cells of old organisms. Thus, critical transcription factors such as E2A and STAT5 show differential expression or activity between young and old B cells. As a result, B-cell physiology appears altered, and antibody production is impaired. Here, we discuss selected phenotypic changes during B-cell aging and attempt to relate them to alterations of molecular mechanisms.

The BCR repertoire comparison, lymphoma typing model and OS predicted model in 5 different pathological lymphomas: T-LBL/ALL, PTCL-NOS, B-MCL, B-FL, and DLBCL.

8059 Background: Lymphatic system cancer is characterized by high heterogeneity in histology and clinical manifestations, B-cell antigen receptor (BCR) plays a vital role in anti-tumor immune responses. This study aimed to compare the BCR repertoire and identify some specific immune markers for different pathological lymphomas. Methods: 5 pathological types of non-Hodgkin's lymphoma (T-LBL/ALL, PTCL-NOS, B-MCL, B-FL, DLBCL) were collected, with reactive lymph node (RLN) hyperplasia as control. All patients were tested by high-throughput immunohistochemical sequencing (HTS-IR) to analyze the correlation between B-cell immunohistochemistry and clinical indicators, and constructed new strategy typing and overall survival (OS) predicted models for lymphomas. Results: The BCR repertoire had the highest diversity in RLN, followed by T-LBL/ALL, PTCL-NOS, DLBCL, B-MCL and B-FL. The diversity of BCR repertoire and similarity of B cell antigens were higher in B-MCL and B-FL patients. Similar to RLN, T-LBL/ALL and PTCL-NOS had broad and diverse V-J pairs, and rare in B-MCL, B-FL and DLBCL. RLN patients were with the highest average number of amino acids, followed by T-LBL/ALL, DLBCL, PTCL-NOS, B-MCL and B-FL. The expressed amino acid sequencing of ARDLIALDY, ARRPGSFDY, ARDIAGWGAVAGLLGRAYYGMDV, and ARDGPYGGNSVEYFQH were markedly different among 5 groups. Patients tended to recurrence expressed ASLDSSPSGFC, ARGMTTVTTAPNY, ARVPLYDDQNINDV and AGGVGGYDWGSYYFDY (P = 0.01605, 0.02869, and 0.01569), and prone to metastasis with expressions of ARVKEFYGILTGYDY, AHSIIGSSWYNWFDP and VRDGGWQSNNWLGFDV (P = 0.04259, 0.0450 and 0.0481). For all patients, 18 (7 negative, 11 positive) and 12 (10 negative, 2 positive) IGH V-J pairs were respectively associated with lymphoma recurrence and metastasis. The top 3 most significant pairs were IGHV7-4-1_IGHJ4, IGHV3-53_IGHJ5 and IGHV3-7_IGHJ5 bound up with recurrence (P = 0.0019, 0.0020 and 0.0021), and IGHV3-74_IGHJ1, IGHV1-69_IGHJ3 and IGHV1-2_IGHJ1 related to metastasis (P = 0.0022, 0.010 and 0.019). The accuracy of typing model in training and test sets was 78.125% (25/32) and 60% (6/10), respectively. The OS model can predict long (≥ 24 months) or short ( < 24 months) OS. Conclusions: Our study identified new biomarkers, constructed novel lymphoma typing model and OS predicted model based on B cell repertoire. It provides a comprehensive understanding of immune response, and contributes to the diagnosis and prognosis of non-Hodgkin's lymphoma.

Cytoskeletal control of the developing and mature B cell antigen receptor repertoire

Abstract The existence of diverse B cell antigen receptor (BCR) specificities is essential for development of protective antibody responses against a wide variety of pathogens. In developing B cells, the BCR repertoire is crafted through sequential rearrangements of heavy and light chain genes, and involves both combinatorial and junctional diversification catalyzed by recombinases and nucleases. Mature B cells undergo further diversification in peripheral lymphoid organs through isotype switching and somatic hypermutations in response to foreign antigens; although these events are not known to alter BCR antigen specificity. Cytoskeletal proteins modulate BCR signal strength and thereby antibody responses, but it is not known if they play a role in the generation of BCR diversity. Using next generation sequencing, and iRepertoire, Inc and ImMunoGeneTics (IMGT) software platforms we examined BCR repertoires in developing and mature B cells that lack the expression of Ezrin, a prominent cytoskeletal regulator of BCR organization, signaling and activation. We show that Ezrin deficiency impacts the generation of BCR repertoire diversity during B cell development, as well as the mature BCR repertoire. Our analysis provides a window into the major processes involved in diversifying the B cell repertoire in control and Ezrin-deficient B cells, including V gene usage, number, frequency and length of unique CDR3s, nucleotide additions and trimming, and shared and unique clonotypes. Our data suggest that the Ezrin cytoskeleton in B cells impacts humoral immunity not only through modulation of B cell activation upon foreign antigen encounter, but also through qualitative and quantitative modulation of the available BCR repertoire.

SHIP interacts with adaptor protein Nck and restricts actin turnover in B cells

SH2 domain-containing inositol 5′-phosphatase (SHIP) has critical functions in regulating signal transduction. In additional to its lipid phosphatase activity, SHIP engages in multiple protein-protein interactions, which can serve to localize either SHIP or its binding partners to a particular subcellular domain. Knock-out and knock-down studies have elucidated that SHIP negatively regulates the accumulation of F-actin in leukocytes, usually resulting in inhibition of actin dependent cellular activities such as spreading and migration. Here, we demonstrate that overexpression of SHIP inhibits B cell antigen receptor (BCR)-mediated cell spreading in murine and human B cell lines. B cell stimulation via the BCR or pervanadate induces an interaction between SHIP and Nck, an adaptor protein known to promote actin polymerization. Using a fluorescence recovery after photobleaching (FRAP) assay, we demonstrate that overexpression of SHIP slows F-actin dynamics in BCR-stimulated B cells and this can be overcome by co-overexpression of Nck. Our data supports a role for SHIP in limiting actin turnover and suggests it may do so in part by sequestering Nck.

Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes.

The major function of B lymphocytes is to sense antigens and to produce protective antibodies after activation. This function requires the expression of a B-cell antigen receptor (BCR), and evolutionary conserved mechanisms seem to exist that ensure that B cells without a BCR do not develop nor survive in the periphery. Here, we show that the loss of BCR expression on Burkitt lymphoma cells leads to decreased mitochondrial function and impaired metabolic flexibility. Strikingly, this phenotype does not result from the absence of a classical Syk-dependent BCR signal but rather from compromised ER expansion. We show that the reexpression of immunoglobulins (Ig) in the absence of the BCR signaling subunits Igα and Igβ rescues the observed metabolic defects. We demonstrate that immunoglobulin expression is needed to maintain ER homeostasis not only in lymphoma cells but also in resting B cells. Our study provides evidence that the expression of BCR components, which is sensed in the ER and shapes mitochondrial function, represents a novel mechanism of metabolic control in B cells.

The role of B cell antigen presentation in the initiation of CD4+ T cell response.

B cells have been known for their ability to present antigens to T cells for almost 40years. However, the precise roles of B cell antigen presentation in various immune responses are not completely understood. The term "professional" antigen-presenting cells (APCs) was proposed to distinguish APCs that are required for initiating the immune responses from those use antigen presentation to enhance their own effector functions. Unlike dendritic cells, which are defined as professional APCs for their well-established functions in activating naive T cells, B cells have been shown in the past to mostly present antigens to activated CD4+ T cells mainly to seek help from T helper cells. However, recent evidence suggested that B cells can act as professional APCs under infectious conditions or conditions mimicking viral infections. B cell antigen receptors (BCRs) and the innate receptor Toll-like receptors are activated synergistically in response to pathogens or virus-like particles, under which conditions B cells are not only potent but also the predominant APCs to turn naive CD4+ T cells into T follicular helper cells. The discovery of B cells as professional APCs to initiate CD4+ T cell response provides a new insight for both autoimmune diseases and vaccine development.

Hide and seek: interplay between influenza viruses and B cells.

Influenza virus constantly acquires genetic mutations/reassortment in the major surface protein, hemagglutinin (HA), resulting in the generation of strains with antigenic variations. There are, however, HA epitopes that are conserved across influenza viruses and are targeted by broadly protective antibodies. A goal for the next-generation influenza vaccines is to stimulate B-cell responses against such conserved epitopes in order to provide broad protection against divergent influenza viruses. Broadly protective B cells, however, are not easily activated by HA antigens with native structure, because the virus has multiple strategies to escape from the humoral immune responses directed to the conserved epitopes. One such strategy is to hide the conserved epitopes from the B-cell surveillance by steric hindrance. Technical advancement in the analysis of the human B-cell antigen receptor (BCR) repertoire has dissected the BCRs to HA epitopes that are hidden in the native structure but are targeted by broadly protective antibodies. We describe here the characterization and function of broadly protective antibodies and strategies that enable B cells to seek these hidden epitopes, with potential implications for the development of universal influenza vaccines.

SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics.

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we develop a method for the case of multiple interconverting species undergoing free diffusion and introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of single-particle trajectory datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.

The Role of Calcium-Calcineurin-NFAT Signaling Pathway in Health and Autoimmune Diseases.

Calcium (Ca2+) is an essential signaling molecule that controls a wide range of biological functions. In the immune system, calcium signals play a central role in a variety of cellular functions such as proliferation, differentiation, apoptosis, and numerous gene transcriptions. During an immune response, the engagement of T-cell and B-cell antigen receptors induces a decrease in the intracellular Ca2+ store and then activates store-operated Ca2+ entry (SOCE) to raise the intracellular Ca2+ concentration, which is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels. Recently, identification of the two critical regulators of the CRAC channel, stromal interaction molecule (STIM) and Orai1, has broadened our understanding of the regulatory mechanisms of Ca2+ signaling in lymphocytes. Repetitive or prolonged increase in intracellular Ca2+ is required for the calcineurin-mediated dephosphorylation of the nuclear factor of an activated T cell (NFAT). Recent data indicate that Ca2+-calcineurin-NFAT1 to 4 pathways are dysregulated in autoimmune diseases. Therefore, calcineurin inhibitors, cyclosporine and tacrolimus, have been used for the treatment of such autoimmune diseases as systemic lupus erythematosus and rheumatoid arthritis. Here, we review the role of the Ca2+-calcineurin–NFAT signaling pathway in health and diseases, focusing on the STIM and Orai1, and discuss the deregulated calcium-mediated calcineurin-NFAT pathway in autoimmune diseases.

Identification of serine residues in the connexin43 carboxyl tail important for BCR-mediated spreading of B-lymphocytes.

B-lymphocytes recognize antigen via B-cell antigen receptors (BCRs). This binding induces signaling, leading to B-cell activation, proliferation and differentiation. Early events of BCR signaling include reorganization of actin and membrane spreading, which facilitates increased antigen gathering. We have previously shown that the gap junction protein connexin43 (Cx43; also known as GJA1) is phosphorylated upon BCR signaling, and its carboxyl tail (CT) is important for BCR-mediated spreading. Here, specific serine residues in the Cx43 CT that are phosphorylated following BCR stimulation were identified. A chimeric protein containing the extracellular and transmembrane domains of CD8 fused to the Cx43 CT was sufficient to support cell spreading. Cx43 CT truncations showed that the region between amino acids 246-307 is necessary for B-cell spreading. Site-specific serine-to-alanine mutations (S255A, S262A, S279A and S282A) resulted in differential effects on both BCR signaling and BCR-mediated spreading. These serine residues can serve as potential binding sites for actin remodeling mediators and/or BCR signaling effectors; therefore, our results may reflect unique roles for each of these serines in terms of linking the Cx43 CT to actin remodeling.

Acalabrutinib and Its Therapeutic Potential in the Treatment of Chronic Lymphocytic Leukemia: A Short Review on Emerging Data.

Recently, the treatment landscape for chronic lymphocytic leukemia (CLL) has changed dramatically due to the development of drugs targeting proteins in the B cell antigen receptor (BCR) pathway. Acalabrutinib, a second-generation Bruton’s tyrosine kinase (BTK) inhibitor, was recently FDA approved for treatment of treatment naïve and relapsed refractory CLL. Acalabrutinib was designed as a more selective BTK inhibitor as compared to ibrutinib in an attempt to mitigate some of the treatment limiting toxicities seen with ibrutinib such as atrial fibrillation and bleeding. In preclinical studies, acalabrutinib was demonstrated to have efficacy in CLL in both patient blood samples and murine models. A multinational phase 1/2 study demonstrated the efficacy and safety of acalabrutinib monotherapy in treatment naïve, relapsed refractory and ibrutinib-intolerant CLL patients. Subsequent phase 3 studies, ASCEND and ELEVATE-TN, compared acalabrutinib monotherapy or combination acalabrutinib and obinutuzumab to standard of care treatments and demonstrated acalabrutinib’s improved efficacy and tolerability. Currently, a phase 3 study is ongoing to compare acalabrutinib to ibrutinib monotherapy (NCT02477696). In the setting of recent FDA approval, real-world evidence will help to elucidate the optimal use of acalabrutinib in the treatment of CLL.

The B-cell antigen receptor of IgE-switched plasma cells regulates memory IgE responses

Allergic inflammation is driven by IgE-producing plasma cells (PCs), which are required for IgE-mediated activation of mast cells and basophils. Repeated antigen encounter elicits a memory IgE response with elevated serum IgE titers and accumulation of IgE-producing PCs. However, the cellular compartment and molecular signals that underlie the immunologic memory of IgE responses remain unclear. With this study we aimed at clarifying whether inactivation of the cytoplasmic immunoglobulin tail tyrosine (ITT) motif in transmembrane IgE (mIgE) impairs the memory IgE response in mice. We generated mice with an inactivated mIgE-ITT motif and analyzed serum IgE levels as well as the generation of IgE-producing germinal center B cells and PCs subsequent to primary and secondary infection with helminths. Invitro cultures were used to study the mIgE-ITT-controlled expression of mIgE on the surface of PCs. Systemic mast cell activation was determined by serum Mcpt1 ELISA in response to ovalbumin challenge. mIgE-ITT-mutant mice showed an impaired memory IgE response subsequent to helminth infection. Furthermore, sensitization and challenge of mIgE-ITT-mutant mice with ovalbumin resulted in diminished serum IgE titers and reduced mast cell activation. The mIgE-ITT motif was required for optimal cell surface expression of mIgE B-cell antigen receptors but not for intracellular IgE expression in PCs. These results indicate that the mIgE B-cell antigen receptor plays a critical role in establishing or maintaining the population of IgE-producing PCs during memory IgE responses.

Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers.

IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

Signal Pathways and Therapeutic Prospects of Diffuse Large B Cell Lymphoma.

Diffuse Large B Cell Lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma which is heterogeneous both clinically and morphologically. Over the past decades, significant advances have been made in the understanding of the molecular genesis, leading to the identification of multiple pathways and molecules that can be targeted for clinical benefit. The current review aims to present a brief overview of signal pathways of DLBCL, which mainly focus on B-cell antigen Receptor (BCR), Nuclear Factor-κB (NF-κB), Phosphatidylinositol-3-Kinase (PI3K) - protein kinase B (Akt) - mammalian Target of Rapamycin (mTOR), Janus Kinase (JAK) - Signal Transducer and Activator (STAT), Wnt/β-catenin, and P53 pathways. Activation of signal pathways may contribute to the generation, development, chemotherapy sensitivity of DLBCL, and expression of pathway molecules is associated with the prognosis of DLBCL. Some agents targeting these pathways have been proved effective and relevant clinical trials are in progress. These agents used single or combined with chemotherapy/each other might raise the possibility of improving clinical outcomes in DLBCL. This review presents several signal pathways of DLBCL and targeted agents had a tendency to improve the curative effect, especially in high-risk or relapsed/refractory DLBCL.

Memory B Cells in Local and Systemic Sites.

Memory B cells are a key cellular component of the protective humoral responses to infectious pathogens. Most of our knowledge of memory B-cell responses comes from studies using mono-epitopic model antigens that elicit systemic humoral responses dominated by canonical B-cell antigen receptors. This approach successfully dissected the systemic responses of memory B cells and greatly advanced our understanding of memory B-cell formation, maintenance, and reactivation to re-invading antigens in the secondary lymphoid organs. However, the canonical memory B-cell responses fail to fully recapitulate the heterogeneity of the protective memory responses. Indeed, accumulating studies using "natural" antigens and live pathogens have uncovered new aspects of memory B-cell responses, which are achieved by memory B cells with different phenotypes, tissue residence, and responsiveness to antigen stimulation. Such non-canonical memory B-cell responses are frequently observed in local sites where live pathogens initially infect and replicate. Importantly, the local memory B-cell responses often serve as the first line of defense against re-infecting pathogens, thereby playing an essential role in controlling the pathogens. Here, we provide a comprehensive overview of the systemic and local memory B-cell responses in the humoral protective immunity against pathogens.

Syk degradation restrains plasma cell formation and promotes zonal transitions in germinal centers.

Germinal centers (GCs) are sites at which B cells proliferate and mutate their antibody-encoding genes in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). B cell antigen receptor (BCR) signals induce Syk activation followed by rapid phosphatase-mediated desensitization; however, how degradation events regulate BCR functions in GCs is unclear. Here, we found that Syk degradation restrains plasma cell (PC) formation in GCs and promotes B cell LZ to DZ transition. Using a mouse model defective in Cbl-mediated Syk degradation, we demonstrate that this machinery attenuates BCR signaling intensity by mitigating the Kras/Erk and PI3K/Foxo1 pathways, and restricting the expression of PC transcription factors in GC B cells. Inhibition of Syk degradation perturbed gene expression, specifically in the LZ, and enhanced the generation of PCs without affecting B cell proliferation. These findings reveal how long-lasting attenuation of signal transduction by degradation events regulates cell fate within specialized microanatomical sites.

Affinity maturation in a human humoral response to influenza hemagglutinin

Affinity maturation of the B cell antigen receptor (BCR) is a conserved and crucial component of the adaptive immune response. BCR lineages, inferred from paired heavy- and light-chain sequences of rearranged Ig genes from multiple descendants of the same naive B cell precursor (the lineages' unmutated common ancestor, "UCA"), make it possible to reconstruct the underlying somatic evolutionary history. We present here an extensive structural and biophysical analysis of a lineage of BCRs directed against the receptor binding site (RBS) of subtype H1 influenza virus hemagglutinin (HA). The lineage includes 8 antibodies detected directly by sequencing, 3 in 1 principal branch and 5 in the other. When bound to HA, the heavy-chain third complementarity determining region (HCDR3) fits with an invariant pose into the RBS, but in each of the 2 branches, the rest of the Fab reorients specifically, from its position in the HA-bound UCA, about a hinge at the base of HCDR3. New contacts generated by the reorientation compensate for contacts lost as the H1 HA mutated during the time between the donor's initial exposure and the vaccination that preceded sampling. Our data indicate that a "pluripotent" naive response differentiated, in each branch, into 1 of its possible alternatives. This property of naive BCRs and persistence of multiple branches of their progeny lineages can offer broader protection from evolving pathogens than can a single, linear pathway of somatic mutation.

A new branched proximity hybridization assay for the quantification of nanoscale protein-protein proximity.

Membrane proteins are organized in nanoscale compartments. Their reorganization plays a crucial role in receptor activation and cell signaling. To monitor the organization and reorganization of membrane proteins, we developed a new branched proximity hybridization assay (bPHA) allowing better quantification of the nanoscale protein-protein proximity. In this assay, oligo-coupled binding probes, such as aptamer, nanobody, and antibodies, are used to translate the proximity of target proteins to the proximity of oligos. The closely positioned oligos then serve as a template for a maximum of 400-fold branched DNA (bDNA) signal amplification. The amplified bPHA signal is recorded by flow cytometer, thus enabling proximity studies with high throughput, multiplexing, and single-cell resolution. To demonstrate the potential of the bPHA method, we measured the reorganization of the immunoglobulin M (IgM)- and immunoglobulin D (IgD)-class B cell antigen receptor (BCR) on the plasma membrane and the recruitment of spleen tyrosine kinase (Syk) to the BCR upon B lymphocyte activation.

Oncogenic Cooperation between EGR1 and BRD4 in Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) grows rapidly and represents 30% to 40% of newly diagnosed lymphomas, and comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). ABC DLBCL is more aggressive with a current cure rate of only ~40%. The B cell antigen receptor (BCR) signaling pathway is known for its critical role in the survival and proliferation of ABC DLBCL cells. Our recent studies revealed that IL6- and IL10-mediated JAK1/STAT3 activation enhances the survival and proliferation of ABC DLBCL cells. Furthermore, we discovered that EGR1, a transcription factor known to be a downstream effector of BCR signaling, is also a downstream target of JAK1 signaling in DLBCL. While EGR1 has been characterized as a tumor suppressor in leukemia, the role of EGR1 in human lymphoma remains unclear. Initial immunohistochemical analysis of 102 DLBCL cases revealed that not only is the level of EGR1 elevated in DLBCL as compared to normal human tonsils, lymph nodes and mantle cell lymphomas, but its level is higher in ABC DLBCL (non-GCB) than GCB DLBCL, indicating functional significance of EGR1 in DLBCL. In addition, we found that 7 out of 8 ABC DLBCL cell lines are sensitive to EGR1 knockdown, whereas only half of 8 GCB DLBCL cell lines are affected. Our genomics studies discovered dual functions of EGR1 in regulating three signaling pathways. EGR1 mediates transcriptional activation associated with two hallmark oncogenic pathways, JAK1/STAT3 and BCR signaling. Our EGR1 ChIP-seq results revealed a large overlap of EGR1 genomic occupancy with that of BRD4, a highly expressed bromo and extraterminal (BET) protein in DLBCL known to induce MYC expression and activation of MYC target genes. This agrees with the RNA-seq results showing that multiple oncogenic pathways including MYC are activated by EGR1 in ABC DLBCL. The synergistic inhibition of cell growth was observed between EGR1 shRNA and JQ1, an inhibitor of BRD4. Surprisingly, the RNA-seq data and pathway analyses also revealed EGR1-mediated suppression of the type I interferon pathway, which otherwise causes cancer cell death. The majority of the interferon pathway genes are up-regulated upon EGR1 knockdown. EGR1-modulated suppression of the interferon pathway appears to be functionally significant as EGR1 knockdown by shRNA synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. EGR1 is known to bind the promoter of p300 and activate p300 expression. P300 catalyzes H3K27 acetylation (H3K27ac), an active histone mark that recruits BRD4 for transcription of MYC and MYC pathway genes, among others. Indeed, immunoblot analysis revealed that EGR1 knockdown reduces H3K27ac expression in ABC DLBCL cells, leading to reduced expression of MYC and MYC target genes based on H3K27ac ChIP qPCR analysis. The results provide a molecular basis for oncogenic cooperation between EGR1 and BRD4 in ABC DLBCL. In summary, our study demonstrates that EGR1 is an oncogenic driver orchestrating multiple important signaling pathways. Notably, EGR1 creates therapeutic vulnerability in ABC DLBCL because both EGR1-mediated transcriptional activation and repression provide growth advantage to the cancer cells. Disclosures No relevant conflicts of interest to declare.