Latent Membrane Protein 1 Signaling Research Articles

Epstein-Barr virus-driven B cell lymphoma mediated by a direct LMP1-TRAF6 complex

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) drives viral B cell transformation and oncogenesis. LMP1’s transforming activity depends on its C-terminal activation region 2 (CTAR2), which induces NF-κB and JNK by engaging TNF receptor-associated factor 6 (TRAF6). The mechanism of TRAF6 recruitment to LMP1 and its role in LMP1 signalling remains elusive. Here we demonstrate that TRAF6 interacts directly with a viral TRAF6 binding motif within CTAR2. Functional and NMR studies supported by molecular modeling provide insight into the architecture of the LMP1-TRAF6 complex, which differs from that of CD40-TRAF6. The direct recruitment of TRAF6 to LMP1 is essential for NF-κB activation by CTAR2 and the survival of LMP1-driven lymphoma. Disruption of the LMP1-TRAF6 complex by inhibitory peptides interferes with the survival of EBV-transformed B cells. In this work, we identify LMP1-TRAF6 as a critical virus-host interface and validate this interaction as a potential therapeutic target in EBV-associated cancer.

Characterization of target gene regulation by the two Epstein-Barr virus oncogene LMP1 domains essential for B-cell transformation.

Epstein-Barr virus (EBV) causes multiple human cancers, including B-cell lymphomas. In cell culture, EBV converts healthy human B-cells into immortalized ones that grow continuously, which model post-transplant lymphomas. Constitutive signaling from two cytoplasmic tail domains of the EBV oncogene latent membrane protein 1 (LMP1) is required for this transformation, yet there has not been systematic analysis of their host gene targets. We identified that only signaling from the membrane proximal domain is required for survival of these EBV-immortalized cells and that its loss triggers apoptosis. We identified key LMP1 target genes, whose abundance changed significantly with loss of LMP1 signals, or that were instead upregulated in response to switching on signaling by one or both LMP1 domains in an EBV-uninfected human B-cell model. These included major anti-apoptotic factors necessary for EBV-infected B-cell survival. Bioinformatics analyses identified clusters of B-cell genes that respond differently to signaling by either or both domains.

New Look of EBV LMP1 Signaling Landscape.

Simple SummaryEpstein-Barr Virus (EBV) infection is associated with various lymphomas and carcinomas as well as other diseases in humans. The transmembrane protein LMP1 plays versatile roles in EBV life cycle and pathogenesis, by perturbing, reprograming, and regulating a large range of host cellular mechanisms and functions, which have been increasingly disclosed but not fully understood so far. We summarize recent research progress on LMP1 signaling, including the novel components LIMD1, p62, and LUBAC in LMP1 signalosome and LMP1 novel functions, such as its induction of p62-mediated selective autophagy, regulation of metabolism, induction of extracellular vehicles, and activation of NRF2-mediated antioxidative defense. A comprehensive understanding of LMP1 signal transduction and functions may allow us to leverage these LMP1-regulated cellular mechanisms for clinical purposes. The Epstein–Barr Virus (EBV) principal oncoprotein Latent Membrane Protein 1 (LMP1) is a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily with constitutive activity. LMP1 shares many features with Pathogen Recognition Receptors (PRRs), including the use of TRAFs, adaptors, and kinase cascades, for signal transduction leading to the activation of NFκB, AP1, and Akt, as well as a subset of IRFs and likely the master antioxidative transcription factor NRF2, which we have gradually added to the list. In recent years, we have discovered the Linear UBiquitin Assembly Complex (LUBAC), the adaptor protein LIMD1, and the ubiquitin sensor and signaling hub p62, as novel components of LMP1 signalosome. Functionally, LMP1 is a pleiotropic factor that reprograms, balances, and perturbs a large spectrum of cellular mechanisms, including the ubiquitin machinery, metabolism, epigenetics, DNA damage response, extracellular vehicles, immune defenses, and telomere elongation, to promote oncogenic transformation, cell proliferation and survival, anchorage-independent cell growth, angiogenesis, and metastasis and invasion, as well as the development of the tumor microenvironment. We have recently shown that LMP1 induces p62-mediated selective autophagy in EBV latency, at least by contributing to the induction of p62 expression, and Reactive Oxygen Species (ROS) production. We have also been collecting evidence supporting the hypothesis that LMP1 activates the Keap1-NRF2 pathway, which serves as the key antioxidative defense mechanism. Last but not least, our preliminary data shows that LMP1 is associated with the deregulation of cGAS-STING DNA sensing pathway in EBV latency. A comprehensive understanding of the LMP1 signaling landscape is essential for identifying potential targets for the development of novel strategies towards targeted therapeutic applications.

Single nucleotide polymorphisms within NFKBIA are associated with nasopharyngeal carcinoma susceptibility in Chinese Han population

Genes involved in latent membrane protein 1 (LMP1) signaling pathways have been suggested to play an important role in nasopharyngeal carcinogenesis. We investigated potentially functional genetic variants associated with the risk of nasopharyngeal carcinoma (NPC) in genes involved in the LMP1 signaling pathway. Altogether, 73 single nucleotide polymorphisms (SNPs) with MAF ≥ 10% were located within the regions of interest of the four genes TRAF3, NFKBIA, CHUK and MAP2K4. From these, 10 SNPs were selected for genotyping based on LD (r2 ≥ 0.80) in a hospital-based case-control study of 332 NPC cases and 585 healthy controls from the Chinese Han population. Minor allele carriers of the promoter SNP rs2233409 in NFKBIA, had an increased risk of NPC (AA vs GG: OR 7.14, 95%CI, 1.08–34.18, P = 0.04, dominant model). Based on the results, we concluded that rs2233409 polymorphism in NFKBIA may be moderately associated with the risk of NPC. Further studies with larger independent samples and functional analysis are needed to verify our results.

A central role of IKK2 and TPL2 in JNK activation and viral B-cell transformation

IκB kinase 2 (IKK2) is well known for its pivotal role as a mediator of the canonical NF-κB pathway, which has important functions in inflammation and immunity, but also in cancer. Here we identify a novel and critical function of IKK2 and its co-factor NEMO in the activation of oncogenic c-Jun N-terminal kinase (JNK) signaling, induced by the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV). Independent of its kinase activity, the TGFβ-activated kinase 1 (TAK1) mediates LMP1 signaling complex formation, NEMO ubiquitination and subsequent IKK2 activation. The tumor progression locus 2 (TPL2) kinase is induced by LMP1 via IKK2 and transmits JNK activation signals downstream of IKK2. The IKK2-TPL2-JNK axis is specific for LMP1 and differs from TNFα, Interleukin−1 and CD40 signaling. This pathway mediates essential LMP1 survival signals in EBV-transformed human B cells and post-transplant lymphoma, and thus qualifies as a target for treatment of EBV-induced cancer.

Epstein-Barr Virus Latent Membrane Protein 1 Regulates Host B Cell MicroRNA-155 and Its Target FOXO3a via PI3K p110α Activation.

Epstein-Barr Virus (EBV) is associated with potentially fatal lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), a serious complication of transplantation. The viral mechanisms underlying the development and maintenance of EBV+ B cell lymphomas remain elusive but represent attractive therapeutic targets. EBV modulates the expression of host microRNAs (miRs), non-coding RNAs that regulate gene expression, to promote survival of EBV+ B cell lymphomas. Here, we examined how the primary oncogene of EBV, latent membrane protein 1 (LMP1), regulates host miRs using an established model of inducible LMP1 signaling. LMP1 derived from the B95.8 lab strain or PTLD induced expression of the oncogene miR-155. However, PTLD variant LMP1 lost the ability to upregulate the tumor suppressor miR-193. Small molecule inhibitors (SMI) of p38 MAPK, NF-κB, and PI3K p110α inhibited upregulation of miR-155 by B95.8 LMP1; no individual SMI significantly reduced upregulation of miR-155 by PTLD variant LMP1. miR-155 was significantly elevated in EBV+ B cell lymphoma cell lines and associated exosomes and inversely correlated with expression of the miR-155 target FOXO3a in cell lines. Finally, LMP1 reduced expression of FOXO3a, which was rescued by a PI3K p110α SMI. Our data indicate that tumor variant LMP1 differentially regulates host B cell miR expression, suggesting viral genotype as an important consideration for the treatment of EBV+ B cell lymphomas. Notably, we demonstrate a novel mechanism in which LMP1 supports the regulation of miR-155 and its target FOXO3a in B cells through activation of PI3K p110α. This mechanism expands on the previously established mechanisms by which LMP1 regulates miR-155 and FOXO3a and may represent both rational therapeutic targets and biomarkers for EBV+ B cell lymphomas.

Delineation of the Viral and Host Cell Genomic Alterations in EBV-positive PTLD

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication of organ transplantation characterized by abnormal proliferation of lymphoid cells in the setting of immunosuppression after transplantation. Epstein-Barr virus (EBV) is responsible for abnormal lymphocyte proliferation in 50–80% of PTLDs, particularly in early-onset disease. Latent membrane protein 1 (LMP1) is the chief oncogenic protein of EBV and has been shown to be critical in the transformation of human B cells by the virus. LMP1 may activate several cellular signal transduction pathways including MAPK/ERK, p38/JNK, PI3K/AKT and NF-kB/AP-1. The ability to usurp and dysregulate signaling pathways may explain how LMP1 promotes cell transformation, survival, and proliferation. Our laboratory has shown that LMP1 isolated from EBV-associated B cell lymphoma lines of PTLD patients contains gain-of-function mutations at AA212 (G-S) and AA366 (S-T) that result in sustained ERK signaling, c-Fos activation, and AP-1 activity. In this study, we asked whether these mutations, or other genetic alterations, are present in primary EBV+ PTLD tumors themselves. DNA was isolated from formalin-fixed paraffin-embedded tissue sections of EBV+ PTLD tumors (n=8). Nested PCR was used to amplify LMP1, and the PCR products generated were cloned and sequenced. The presence or absence of gain-of-function mutations at AA212 (G-S) and AA366 (S-T) were assessed. 7 out of the 8 EBV+ PTLD tumors demonstrated both gain-of-function mutations in LMP1. Furthermore, 6 out of the 8 tumors contained an extra repeat of 8 amino acids within the LMP1 signaling tail corresponding to a putative JAK3 binding motif. We have previously identified this repeat in 3 of 6 EBV+ B cell lymphoma lines from PTLD patients. LMP1 was also cloned from the whole blood of a pediatric small bowel PTLD patient. In addition to these 2 mutations being present, the 8 amino acid repeat was also present but in triplicate, suggesting this motif may be crucial to the oncogenic activity of LMP1. In order to assess host cell mutations in EBV+ PTLD tumors, a qBiomarker Mutation PCR array was performed for the PI3K/AKT/mTOR pathway, known to be important in human malignancies. While mutations in PI3K/AKT/mTOR were identified in EBV+ B cell lymphoma lines from PTLD patients, there were significantly more mutations in the primary EBV+ PTLD tumors, with 25 distinct mutations found within the PTEN, PI3K, and STK11 molecules. 3 distinct mutations were identified in more than 1 tumor, and 4 distinct mutations were shared between a cell line and tumor. Our findings clearly demonstrate that key gain-of-functions mutations in LMP1 detected in blood and cell lines are also detected in the primary tumor, suggesting a role in tumorigenesis and great potential as biomarkers of EBV+ PTLD. Moreover, host cell mutations may also contribute to dysregulation of key signal transduction pathways in EBV+ PTLD.

LIMD1 is induced by and required for LMP1 signaling, and protects EBV-transformed cells from DNA damage-induced cell death.

LIMD1 (LIM domain-containing protein 1) is considered as a tumor suppressor, being deregulated in many cancers to include hematological malignancies; however, very little is known about the underlying mechanisms of its deregulation and its roles in carcinogenesis. Epstein-Barr Virus (EBV) is associated with a panel of malignancies of lymphocytic and epithelial origin. Using high throughput expression profiling, we have previously identified LIMD1 as a common marker associated with the oncogenic transcription factor IRF4 in EBV-related lymphomas and other hematological malignancies. In this study, we have identified potential conserved IRF4- and NFκB-binding motifs in the LIMD1 gene promoter, and both are demonstrated functional by promoter-reporter assays. We further show that LIMD1 is partially upregulated by EBV latent membrane protein 1 (LMP1) via IRF4 and NFκB in EBV latency. As to its role in the setting of EBV latent infection, we show that LIMD1 interacts with TRAF6, a crucial mediator of LMP1 signal transduction. Importantly, LIMD1 depletion impairs LMP1 signaling and functions, potentiates ionomycin-induced DNA damage and apoptosis, and inhibits p62-mediated selective autophagy. Taken together, these results show that LIMD1 is upregulated in EBV latency and plays an oncogenic role rather than that of a tumor suppressor. Our findings have identified LIMD1 as a novel player in EBV latency and oncogenesis, and open a novel research avenue, in which LIMD1 and p62 play crucial roles in linking DNA damage response (DDR), apoptosis, and autophagy and their potential interplay during viral oncogenesis.

N-acetylcysteine (NAC) ameliorates Epstein-Barr virus latent membrane protein 1 induced chronic inflammation.

Chronic inflammation results when the immune system responds to trauma, injury or infection and the response is not resolved. It can lead to tissue damage and dysfunction and in some cases predispose to cancer. Some viruses (including Epstein-Barr virus (EBV)) can induce inflammation, which may persist even after the infection has been controlled or cleared. The damage caused by inflammation, can itself act to perpetuate the inflammatory response. The latent membrane protein 1 (LMP1) of EBV is a pro-inflammatory factor and in the skin of transgenic mice causes a phenotype of hyperplasia with chronic inflammation of increasing severity, which can progress to pre-malignant and malignant lesions. LMP1 signalling leads to persistent deregulated expression of multiple proteins throughout the mouse life span, including TGFα S100A9 and chitinase-like proteins. Additionally, as the inflammation increases, numerous chemokines and cytokines are produced which promulgate the inflammation. Deposition of IgM, IgG, IgA and IgE and complement activation form part of this process and through genetic deletion of CD40, we show that this contributes to the more tissue-destructive aspects of the phenotype. Treatment of the mice with N-acetylcysteine (NAC), an antioxidant which feeds into the body’s natural redox regulatory system through glutathione synthesis, resulted in a significantly reduced leukocyte infiltrate in the inflamed tissue, amelioration of the pathological features and delay in the inflammatory signature measured by in vivo imaging. Reducing the degree of inflammation achieved through NAC treatment, had the knock on effect of reducing leukocyte recruitment to the inflamed site, thereby slowing the progression of the pathology. These data support the idea that NAC could be considered as a treatment to alleviate chronic inflammatory pathologies, including post-viral disease. Additionally, the model described can be used to effectively monitor and accurately measure therapies for chronic inflammation.

Epstein-Barr Virus LMP1-Mediated Oncogenicity.

Epstein-Barr virus latent membrane protein 1 (LMP1) is expressed in multiple human malignancies, including nasopharyngeal carcinoma and Hodgkin and immunosuppression-associated lymphomas. LMP1 mimics CD40 signaling to activate multiple growth and survival pathways, in particular, NF-κB. LMP1 has critical roles in Epstein-Barr virus (EBV)-driven B-cell transformation, and its expression causes fatal lymphoproliferative disease in immunosuppressed mice. Here, we review recent developments in studies of LMP1 signaling, LMP1-induced host dependency factors, mouse models of LMP1 lymphomagenesis, and anti-LMP1 immunotherapy approaches.

Expression of interferon regulatory factor 7 correlates with the expression of Epstein-Barr Virus latent membrane protein 1 and cervical lymph node metastasis in nasopharyngeal cancer.

Interferon regulatory factor 7 (IRF7) has oncogenic properties in several malignancies such as Epstein-Barr virus (EBV)-associated lymphoma. However, there is no evidence whether IRF7 is associated with the oncogenesis of nasopharyngeal cancer (NPC), the pathogenesis of which is closely associated with EBV. Herein, we report that expression of IRF7 was increased in normal nasopharyngeal cells that expressed the EBV principal oncoprotein, latent membrane protein 1 (LMP1). In addition, IRF7 was mainly expressed in the nucleus in both normal nasopharyngeal cells and nasopharyngeal cancer cells that expresses LMP1. On immunohistochemical analysis, IRF7 was predominantly localized in the nucleus in biopsy samples of NPC tissues. In total, IRF7 expression was detected with 36 of 49 specimens of these tissues. Furthermore, the expression score of IRF7 correlated with the expression score of LMP1. Moreover, the expression score of IRF7 is associated with cervical lymph-node metastasis, which reflects the highly metastatic nature of this cancer. Taken together, our results suggest that expression of IRF7 is one of the metastatic effectors of LMP1 signalling in EBV-associated NPC.

PD-L1 Is up-Regulated By EBV-Driven LMP1 through NF-κb Pathway and Correlates with Poor Prognosis in Natural Killer/T-Cell Lymphoma

Background:Natural killer/T-cell lymphoma (NKTCL) is an EBV-associated, highly aggressive lymphoma. Treatment outcome remains sub-optimal, especially for advanced-stage or relapsed diseases. Programmed cell death receptor 1 (PD-1) and PD ligand 1 (PD-L1) have become promising therapeutic targets for various malignancies, but their role in the pathogenesis and their interactions with Epstein-Barr virus (EBV) in NKTCL remains to be investigated.Materials and methods:Expression of PD-L1 was measured in NK-92 (EBV-negative) and SNK-6 (EBV-positive) cells by western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay, respectively. Latent membrane protein 1 (LMP1)-harboring lentiviral vectors were transfected into NK-92 cells to examine the correlation between LMP1 and PD-L1 expression. Proteins in the downstream pathways of LMP1 signaling were measured in NK-92 cells transfected with LMP1-harboring or negative control vectors, respectively. PD-L1 expression on tumor specimens and serum concentration of soluble PD-L1 were collected in a retrospective cohort of patients with Ann Arbor stage I~II NKTCL and their prognostic significance were analyzed.Results:Expression of PD-L1 was significantly higher in SNK-6 cells than in NK-92 cells, at both protein and mRNA levels. Expression of PD-L1was remarkably up-regulated in NK-92 cells transfected with LMP1-harboring lentiviral vectors compared with those transfected with negative control vectors. Proteins in the MAPK/NF-κB pathway were up-regulated in LMP1-expressing NK-92 cells compared with the negative control. Selective inhibitors of those proteins induced significant down-regulation of PD-L1 expression in LMP1-expressing NK-92 cells. Patients with a high concentration of serum soluble PD-L1 (≥ 3.4 ng/ml) or with a high percentage of PD-L1 expression in tumor specimens (≥ 38%) exhibited significantly lower response rate to treatment and remarkably worse survival, compared with their counterparts. A high concentration of serum soluble PD-L1 and a high percentage of PD-L1 expression in tumor specimens were independent adverse prognostic factors among patients with stage I~II NKTCL.Conclusions:PD-L1 expression positively correlated LMP1 expression in NKTCL, which was probably mediated by the MAPK/NF-κB pathway. PD-L1 expression in serum and tumor tissues have significant prognostic value for early-stage NKTCL. DisclosuresNo relevant conflicts of interest to declare.

LMP1 signaling pathway activates IRF4 in latent EBV infection and a positive circuit between PI3K and Src is required.

Interferon (IFN) regulatory factors (IRFs) have crucial roles in immune regulation and oncogenesis. We have recently shown that IRF4 is activated through c-Src-mediated tyrosine phosphorylation in virus-transformed cells. However, the intracellular signaling pathway triggering Src activation of IRF4 remains unknown. In this study, we provide evidence that Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) promotes IRF4 phosphorylation and markedly stimulates IRF4 transcriptional activity, and that Src mediates LMP1 activation of IRF4. As to more precise mechanism, we show that LMP1 physically interacts with c-Src, and the phosphatidylinositol 3 kinase (PI3K) subunit P85 mediates their interaction. Depletion of P85 by P85-specific short hairpin RNAs disrupts their interaction and diminishes IRF4 phosphorylation in EBV-transformed cells. Furthermore, we show that Src is upstream of PI3K for activation of both IRF4 and Akt. In turn, inhibition of PI3K kinase activity by the PI3K-speicfic inhibitor LY294002 impairs Src activity. Our results show that LMP1 signaling is responsible for IRF4 activation, and further characterize the IRF4 regulatory network that is a promising therapeutic target for specific hematological malignancies.

PD-L1 is upregulated by EBV-driven LMP1 through NF-κB pathway and correlates with poor prognosis in natural killer/T-cell lymphoma.

BackgroundNatural killer/T-cell lymphoma (NKTCL) is an Epstein–Barr virus (EBV)-associated, highly aggressive lymphoma. Treatment outcome remains sub-optimal, especially for advanced-stage or relapsed diseases. Programmed cell death receptor 1 (PD-1) and PD ligand 1 (PD-L1) have become promising therapeutic targets for various malignancies, but their role in the pathogenesis and their interactions with EBV in NKTCL remains to be investigated.MethodsExpression of PD-L1 was measured in NK-92 (EBV-negative) and SNK-6 (EBV-positive) cells by western blot, quantitative real-time PCR and enzyme-linked immunosorbent assay, and flow cytometry, respectively. Latent membrane protein 1 (LMP1)-harboring lentiviral vectors were transfected into NK-92 cells to examine the correlation between LMP1 and PD-L1 expression. Proteins in the downstream pathways of LMP1 signaling were measured in NK-92 cells transfected with LMP1-harboring or negative control vectors as well as in SNK-6 cells. PD-L1 expression on tumor specimens and serum concentration of soluble PD-L1 were collected in a retrospective cohort of patients with Ann Arbor stage I~II NKTCL, and their prognostic significance were analyzed.ResultsExpression of PD-L1 was significantly higher in SNK-6 cells than in NK-92 cells, at both protein and mRNA levels. Expression of PD-L1 was remarkably upregulated in NK-92 cells transfected with LMP1-harboring lentiviral vectors compared with those transfected with negative control vectors. Proteins in the MAPK/NF-κB pathway were upregulated in LMP1-expressing NK-92 cells compared with the negative control. Selective inhibitors of those proteins induced significant downregulation of PD-L1 expression in LMP1-expressing NK-92 cells as well as in SNK-6 cells. Patients with a high concentration of serum soluble PD-L1 (≥3.4 ng/ml) or with a high percentage of PD-L1 expression in tumor specimens (≥38 %) exhibited significantly lower response rate to treatment and remarkably worse survival, compared with their counterparts. A high concentration of serum soluble PD-L1 and a high percentage of PD-L1 expression in tumor specimens were independent adverse prognostic factors among patients with stage I~II NKTCL.ConclusionsPD-L1 expression positively correlated LMP1 expression in NKTCL, which was probably mediated by the MAPK/NF-κB pathway. PD-L1 expression in serum and tumor tissues has significant prognostic value for early-stage NKTCL.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0341-7) contains supplementary material, which is available to authorized users.

Regulation of IgD Expression and Its Role in B Cell Transformation

A developing B lymphocyte first acquires expression of immunoglobulin M (IgM) at the immature stage, and along maturation into the transitional stage it starts to co-express another isotype-IgD. IgM and IgD are generated by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus, so that they share the N-terminal antigen-binding variable region, but differ in the C-terminal constant region, encoded by Cμ or Cδ exons, respectively. Maturation into follicular (naïve) B cells, IgD levels surpass those of IgM; while upon antigen-stimulation and entering germinal center reaction, follicular B cells turn off IgD expression. Trans-acting factors have been proposed to control IgM and IgD expression since several decades ago. However, the first of such factors, Zfp318, was just reported last year (Enders et al. Proc Natl Acad Sci USA 2014; Pioli et al. J Immunol 2014). Still, other factors and signaling cascades regulating IgD expression remain to be identified.In this context, we accidentally discovered that expression of the Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) in B cells specifically abolishes IgD but not IgM expression; LMP1 signaling suppresses IgD expression at the mRNA level. Using microarray we screened differentially expressed genes in LMP1-expressing versus normal mouse B cells, and then looked up these genes in ImmuGen database (http://www.immgen.org) to identify those whose expression patterns correlate with that of IgD at various B cell developmental stages. These analyses returned us two new candidates Rapgef4 and CD55, in addition to Zfp318. By CRISPR technology, we further demonstrated that mutation of Rapgef4 or CD55 in mature B cells results in downregulation of IgD but not IgM. To define their role in regulating IgD expression in vivo, mice carrying conditional null alleles Rapgef4F/F and CD55F/F, are being generated.The facts that IgD is an evolutionarily conserved Ig isotype and its expression is precisely regulated during B cell developmental and functional stages suggest an important role in B cell biology/function. Indeed, a recent study suggested that IgD, by sequestering CD19 coreceptor from IgM, might play a negative role in B cell receptor (BCR) signaling (Klasener et al. eLife 2014). Interestingly, flow cytometric analysis of 12 diffuse large B cell lymphoma (DLBCL)-like malignancies arising in our mouse model revealed that 10 cases are low/negative for IgD and the other 2 cases express intermediate levels of IgD, while they all express surface IgM or IgG (Zhang et al. Cell Rep 2015; data not shown); human DLBCLs often retain surface IgM expression (Wright et al. Proc Natl Acad Sci USA 2003; IgD expression data not available). Basing on aforementioned observations, we hypothesize that downregulation of IgD may be important for LMP1-induced B cell transformation and for DLBCL pathogenesis in general. Accordingly, in this work, we will reconstitute IgD expression in LMP1+ lymphoma cell lines (Zhang et al. Cell 2012), and determine if it suppresses lymphoma cell growth in vitro and in vivo. Similarly, we will reconstitute IgD expression in a few IgD- mouse DLBCL lines and monitor its impact on lymphoma growth. In addition, as we have 2 DLBCL lines expressing intermediate levels of IgD, we will use CRISPR technology to delete/mutate Cδ coding sequence in these lymphoma lines, and see if the mutated cells (losing IgD expression) outcompete the non-mutated cells.Overall, this work will lead to molecular insights into IgD regulation. It may also elucidate the functional significance of IgD downregulation in B cell lymphoma pathogenesis/maintenance, and guide therapy development targeting BCR signaling. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Latent Membrane Protein 1 Signaling through Interaction with Cytoskeletal Proteins.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) induces constitutive signaling in EBV-infected cells to ensure the survival of the latently infected cells. LMP1 is localized to lipid raft domains to induce signaling. In the present study, a genome-wide screen based on bimolecular fluorescence complementation (BiFC) was performed to identify LMP1-binding proteins. Several actin cytoskeleton-associated proteins were identified in the screen. Overexpression of these proteins affected LMP1-induced signaling. BiFC between the identified proteins and LMP1 was localized to lipid raft domains and was dependent on LMP1-induced signaling. Proximity biotinylation assays with LMP1 induced biotinylation of the actin-associated proteins, which were shifted in molecular mass. Together, the findings of this study suggest that the association of LMP1 with lipid rafts is mediated at least in part through interactions with the actin cytoskeleton. LMP1 signaling requires oligomerization, lipid raft partitioning, and binding to cellular adaptors. The current study utilized a genome-wide screen to identify several actin-associated proteins as candidate LMP1-binding proteins. The interaction between LMP1 and these proteins was localized to lipid rafts and dependent on LMP1 signaling. This suggests that the association of LMP1 with lipid rafts is mediated through interactions with actin-associated proteins.

Changes in expression induced by Epstein-Barr Virus LMP1-CTAR1: potential role of bcl3.

ABSTRACTThe Epstein-Barr virus protein latent membrane protein 1 (LMP1) has two NF-κB activating domains within its intracellular carboxy terminus (carboxy-terminal activating region 1 [CTAR1] and CTAR2). LMP1-CTAR1 is required for B-lymphocyte transformation, is capable of transforming rodent fibroblasts, and uniquely activates phosphoinositol (PI3) kinase, the noncanonical NF-κB pathway, and expression of the epidermal growth factor receptor (EGFR). In this study, the effects of LMP1-CTAR1 on cellular gene expression were determined by high-throughput sequencing. Additionally, the binding of bcl3 was determined using chromatin immunoprecipitation (ChIP) and sequencing. LMP1-CTAR1 induced few changes in transcription with more genes showing decreased expression. Ingenuity pathway analysis indicated significant enrichment for genes involved in cancer and cellular movement, survival, growth, and proliferation pathways. ChIP in combination with high-throughput sequencing (ChIP-Seq) identified bcl3 binding for more than 2,000 genes in LMP1-CTAR1-expressing cells with more than 90% of the peaks at genes detected within the probable promoter region. Only a small subset of the genes with significant changes in expression had corresponding peaks in the bcl3 ChIP. However, both NFKB2 and PI3 kinase were identified in the bcl3 ChIP. Additionally, many of the predicted upstream regulators for the changes in expression were identified in the bcl3 ChIP. Analysis of the proteins in the NF-κB pathway revealed many changes identified by the high-throughput RNA sequencing (RNA-Seq) and bcl3 ChIP that would likely activate noncanonical NF-κB signaling and possibly inhibit canonical NF-κB signaling. These findings suggest that the two LMP1 signaling domains modulate their combined activity and that the bcl3 transcription factor is likely responsible for some of the unique effects of CTAR1 on cellular expression.

Novel roles and therapeutic targets of Epstein-Barr virus-encoded latent membrane protein 1-induced oncogenesis in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) was first discovered 50 years ago as an oncogenic gamma-1 herpesvirus and infects more than 90% of the worldwide adult population. Nasopharyngeal carcinoma (NPC) poses a serious health problem in southern China and is one of the most common cancers among the Chinese. There is now strong evidence supporting a role for EBV in the pathogenesis of NPC. Latent membrane protein 1 (LMP1), a primary oncoprotein encoded by EBV, alters several functional and oncogenic properties, including transformation, cell death and survival in epithelial cells in NPC. LMP1 may increase protein modification, such as phosphorylation, and initiate aberrant signalling via derailed activation of host adaptor molecules and transcription factors. Here, we summarise the novel features of different domains of LMP1 and several new LMP1-mediated signalling pathways in NPC. When then focus on the potential roles of LMP1 in cancer stem cells, metabolism reprogramming, epigenetic modifications and therapy strategies in NPC.

Exosomes derived from Burkitt's lymphoma cell lines induce proliferation, differentiation, and class-switch recombination in B cells.

Exosomes, nano-sized membrane vesicles, are released by various cells and are found in many human body fluids. They are active players in intercellular communication and have immune-suppressive, immune-regulatory, and immune-stimulatory functions. EBV is a ubiquitous human herpesvirus that is associated with various lymphoid and epithelial malignancies. EBV infection of B cells in vitro induces the release of exosomes that harbor the viral latent membrane protein 1 (LMP1). LMP1 per se mimics CD40 signaling and induces proliferation of B lymphocytes and T cell-independent class-switch recombination. Constitutive LMP1 signaling within B cells is blunted through the shedding of LMP1 via exosomes. In this study, we investigated the functional effect of exosomes derived from the DG75 Burkitt's lymphoma cell line and its sublines (LMP1 transfected and EBV infected), with the hypothesis that they might mimic exosomes released during EBV-associated diseases. We show that exosomes released during primary EBV infection of B cells harbored LMP1, and similar levels were detected in exosomes from LMP1-transfected DG75 cells. DG75 exosomes efficiently bound to human B cells within PBMCs and were internalized by isolated B cells. In turn, this led to proliferation, induction of activation-induced cytidine deaminase, and the production of circle and germline transcripts for IgG1 in B cells. Finally, exosomes harboring LMP1 enhanced proliferation and drove B cell differentiation toward a plasmablast-like phenotype. In conclusion, our results suggest that exosomes released from EBV-infected B cells have a stimulatory capacity and interfere with the fate of human B cells.

A new model of LMP1–MYC interaction in B cell lymphoma

Epstein–Barr virus (EBV) is associated with aggressive B cell lymphomas (BCLs). Latent membrane protein 1 (LMP1) of EBV is an oncogenic protein required for EBV B cell transformation. However, LMP1 is a weak oncogene in mice. Mice expressing Myc inserted 5’ of the Eμ enhancer (iMycEμ), mimicking the t(8;14) translocation of endemic Burkitt lymphoma, develop delayed onset BCLs. To investigate potential cooperation between LMP1 and oncogenic MYC, we produced mice expressing the LMP1 signaling domain via a hybrid CD40–LMP1 transgene (mCD40–LMP1), and the dysregulated MYC protein of aggressive EBV+ BCLs. mCD40-LMP1/iMycEμ mice trended toward earlier BCL onset. BCLs from mCD40–LMP1/iMycEμ mice expressed LMP1 and were transplantable into immunocompetent recipients. iMycEμ and mCD40–LMP1/iMycEμ mice developed BCLs with similar immunophenotypes. LMP1 signaling was intact in BCLs as shown by inducible interleukin-6. Additionally, LMP1 signaling to tumor cells induced the two isoforms of Pim1, a constitutively active prosurvival kinase implicated in lymphomagenesis.