Nathalie Faumont

c-Myc and Rel/NF-kappaB are the two master transcriptional systems activated in the latency III program of Epstein-Barr virus-immortalized B cells.

ABSTRACT The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-κB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-κB are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV.

Human Monocytic Cell Lines Transformed In Vitro by Epstein-Barr Virus Display a Type II Latency and LMP-1-Dependent Proliferation

ABSTRACT Epstein-Barr virus (EBV) classically infects and transforms B lymphocytes in vitro, yielding lymphoblastoid cell lines (LCLs). In contrast to other herpesviruses, EBV is not described as an infectious agent for monocytes. However, recent papers described in vitro infection of monocytes leading to abortive or transient viral expression. In the present study, we report the characterization of E1, a monocytic cell line infected and transformed by EBV. This cell line was derived from an LCL by a drastic electroporation and selection of neomycin-resistant cells, unfavorable to B-cell outgrowth. E1 expressed surface molecules of monocytic lineage (CD14, major histocompatibility complex class II, and CD80) and the c-fms gene, a highly specific marker for the monocytic lineage. This cell line is able to phagocytose and secrete proinflammatory monokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. E1 cells are tumorigenic after injection in nude mice, and a monocytic cell line obtained from one of these tumors (TE1) displayed immunophenotype and functional properties similar to those of E1. We detected the presence of the EBV genome in both cell lines, as well as expression of the EBNA-1 and LMP-1, but not EBNA-2, viral genes, characteristic of a type II latency. LMP-1 influences the phenotype of these monocytic cell lines, as demonstrated by down-regulation of cell proliferation and membrane intercellular adhesion molecule 1 expression due to an LMP-1 antisense strategy. This is the first description of a latently infected human monocytic cell line and the first direct demonstration of an instrumental role for LMP-1 in the proliferation of EBV-transformed cell lines expressing a type II latency.

Evidence for early infection of nonneoplastic natural killer cells by Epstein-Barr virus.

ABSTRACT We examined lymph nodes and tonsils from patients with infectious mononucleosis by combined detection of EBV-encoded RNA and a specific marker of natural killer (NK) cells, PEN5. A small number of Epstein-Barr virus (EBV) latently infected nonneoplastic NK cells were detected. Our data demonstrate that NK cells are natural targets of EBV and that infection of these cells is an early event observed during primary EBV infection.

Development of a conditional bioluminescent transplant model for TPM3-ALK-induced tumorigenesis as a tool to validate ALK-dependent cancer targeted therapy.

Overexpression and activation of TPM3-ALK tyrosine kinase fusion protein is a causal oncogenic event in the development of Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic ALK-positive tumours. Thus, the development of ALK specific tyrosine kinase inhibitors is a current therapeutic challenge. Animal models are essential to assess, in vivo, the efficiency of ALK-oncogene inhibitors and to identify new and/or additional therapeutic targets in the ALK tumorigenesis pathway. Using the tetracycline system to allow conditional and concomitant TPM3-ALK and luciferase expression, we have developed a unique transplant model for bioluminescent TPM3-ALK-induced fibroblastic tumours in athymic nude mice. The reversible TPM3-ALK expression allowed us to demonstrate that this oncogene is essential for the tumour growth and its maintenance. In addition, we showed that this model could be used to precisely assess tumour growth inhibition upon ALK chemical inactivation. As proof of principle, we used the general tyrosine kinase inhibitor herbimycin A to inhibit ALK oncoprotein activity. As expected, herbimycin A treatment reduced tumour growth as assessed both by tumour volume measurement and bioluminescent imaging. We conclude that this transplant model for TPM3-ALK-induced tumours represents a valuable tool not only to accurately and rapidly evaluate in vivo ALK-targeted therapies but also to gain insight into the mechanism of ALK-positive tumour development.

B7-H1, Which Represses EBV-Immortalized B Cell Killing by Autologous T and NK Cells, Is Oppositely Regulated by c-Myc and EBV Latency III Program at Both mRNA and Secretory Lysosome Levels

EBV-immortalized B cells induce a complex immune response such that the virus persists as a clinically silent infection for the lifetime of the infected host. B7-H1, also called PD-L1, is a cosignaling molecule of the B7 family that can inhibit activated T cell effectors by interaction with its receptor PD-1. In this work, we have studied the dependence of B7-H1 on NF-κB and c-Myc, the two main transcription factors in EBV latency III proliferating B cells, on various lymphoblastoid and Burkitt lymphoma cell lines, some of them being inducible or not for the EBV latency III program and/or for c-Myc. We found that B7-H1 repressed killing of EBV-immortalized B cells by their autologous T and NK cells. At the mRNA level, NF-κB was a weak inducer whereas c-Myc was a strong repressor of B7-H1 expression, an effect mediated by STAT1 inhibition. At the protein level, B7-H1 molecules were stored in both degradative and unconventional secretory lysosomes. Surface membrane B7-H1 molecules were constitutively internalized and proteolyzed in lysosomes. The EBV latency III program increased the amounts of B7-H1–containing secretory lysosomes and their export to the surface membrane. By repressing actin polymerization, c-Myc blocked secretory lysosome migration and B7-H1 surface membrane export. In addition to B7-H1, various immunoregulatory molecules participating in the immunological synapse are stored in secretory lysosomes. By playing on actin polymerization, c-Myc could thus globally regulate the immunogenicity of transformed B cells, acting on export of secretory lysosomes to plasma membrane.

Regulation of DNA Polymerase β by the LMP1 Oncoprotein of EBV through the Nuclear Factor-κB Pathway

The repair DNA polymerase beta (Polbeta), when overexpressed, plays a critical role in generating genetic instability via its interference with the genomic replication program. Up-regulation of Polbeta has been reported in many tumor types that exhibit genetic aberrations, including EBV-related B-cell lymphomas. However, the mechanisms responsible for its overexpression have never been examined. Here, we report that both expression and activity of Polbeta, in EBV-immortalized B cells, are induced by several natural genetic variants of LMP1, an oncoprotein associated with the vast majority of EBV-related tumors. Conversely, we found that the expression of Polbeta decreased when LMP1 signaling was down-regulated by a dominant negative of LMP1 or an inhibitor of the nuclear factor-kappaB (NF-kappaB) pathway, the main transduction pathway activated by LMP1, strongly supporting a role of NF-kappaB in the LMP1-mediated Polbeta regulation. Using electrophoretic mobility shift assay experiments from several EBV-immortalized B-cell nuclear extracts, we identified an LMP1-dependent p50/c-Rel heterodimer on a proximal kappaB binding site (-211 to -199nt) of the Polbeta promoter. This result was correlated with a specific Polbeta kappaB transcriptional activity. Taken together, our data enlighten a new mechanism responsible for Polbeta overexpression in EBV-infected cells, mediated by LMP1 and dependent on NF-kappaB activation.

RelA and RelB cross-talk and function in Epstein-Barr virus transformed B cells.

In this study, we determined the respective roles of RelA and RelB NF-κB subunits in Epstein–Barr virus (EBV)-transformed B cells. Using different EBV-immortalized B-cell models, we showed that only RelA activation increased both survival and cell growth. RelB activity was induced secondarily to RelA activation and repressed RelA DNA binding by trapping the p50 subunit. Reciprocally, RelA activation repressed RelB activity by increasing expression of its inhibitor p100. To search for such reciprocal inhibition at the transcriptional level, we studied gene expression profiles of our RelA and RelB regulatable cellular models. Ten RelA-induced genes and one RelB-regulated gene, ARNTL2, were repressed by RelB and RelA, respectively. Apart from this gene, RelB signature was included in that of RelA Functional groups of RelA-regulated genes were for control of energy metabolism, genetic instability, protection against apoptosis, cell cycle and immune response. Additional functions coregulated by RelA and/or RelB were autophagy and plasma cell differentiation. Altogether, these results demonstrate a cross-inhibition between RelA and RelB and suggest that, in fine, RelB was subordinated to RelA. In the view of future drug development, RelA appeared to be pivotal in both classical and alternative activation pathways, at least in EBV-transformed B cells.