Leukemia (2014) 28, 871–879 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE RelA and RelB cross-talk and function in Epstein–Barr virus transformed B cells A Chanut1, F Duguet1, A Marfak1,5, A David1, B Petit2, M Parrens3, S Durand-Panteix1, M Boulin-Deveza1, N Gachard1, I Youlyouz-Marfak1, D Bordessoule1,4, J Feuillard1 and N Faumont1 In this study, we determined the respective roles of RelA and RelB NF-kB 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. Leukemia (2014) 28, 871–879; doi:10.1038/leu.2013.274 Keywords: EBV; NF-kB; cell cycle; metabolism; B-cell lymphoma. INTRODUCTION Two NF-kB activation pathways have been described, so-called Epstein–Barr virus (EBV) is responsible for immunodeficiency- classical (or canonical) and alternative (or non-canonical) 4 related diffuse large B-cell lymphomas (DLBCLs) of posttrans- (Vallabhapurapu and Karin for review). The classical pathway is plant or human immunodeficiency virus-infected patients. EBV is induced in response to a variety of stimuli, such as CD40-Ligand, also associated with various other cancers, including DLBCLs of TNFa, IL-1, IL-6, bacterial lipopolysaccharide, as well as LMP1 of EBV. the elderly, Burkitt, Hodgkin’s or T-cell lymphomas and It involves RelA- or c-Rel- and p50-containing complexes. In resting nasopharyngeal carcinomas.1 In vitro,EBVinfectsand cells, NF-kB dimers containing these subunits are retained in the transforms primary B cells, leading to the continuous cytoplasm by physical interaction with IkBa, b, e or the p105 proliferation of lymphoblastoid cell lines. This proliferation precursor of p50. Following activation of the classical NF-kB program, also called latency III program, is driven by the pathway, the IkBs and p105 are rapidly phosphorylated by the IkB EBNA2 (Epstein–Barr Nuclear Antigen 2) protein, which regulates kinase complex (IKK), containing the catalytically active kinases IKKa expression of the entire set of EBV latent genes, including the and IKKb and the regulatory scaffold protein NEMO (NF-kBEssential BNLF1 gene coding for the main EBV oncogene, the latent Modulator or IKKg).5 Phosphorylation of IkBs leads to their membrane protein 1 (LMP1). Other EBV latent proteins are proteasomal degradation, releasing NF-kB dimers that translocate EBNA1, required for episomal maintenance of the EBV genome, into the nucleus where they activate transcription of specific target the EBNA3 proteins that modulate EBNA2 and regulate cell genes.4 With much slower kinetics, the alternative NF-kB activation proliferation and LMP2A that mimics the B-cell receptor. Some pathway is induced by a restricted subset of receptors such EBV-associated B-cell lymphomas such as endemic Burkitt as the Lymphotoxin b receptor, B-cell-activating factor receptor or lymphomas or the rare primary effusion and plasmaplastic CD40, which then leads to NF-kB-inducing kinase activation.6 lymphomas express only the EBNA1 (so-called latency I). NF-kB-inducing kinase phosphorylates IKKa, which in turn However, most EBV-associated tumors express LMP1, even in mediates phosphorylation and proteolysis of p100, the precursor latency III or II (expression of EBNA1 and LMP proteins). LMP1 is of p52. P100 acts as an IkB molecule specifically trapping a transmembrane protein acting as a constitutive active CD40 RelB-containing complexes (i.e., RelB/p50 and RelB/p52 dimers). receptor, thereby continuously activating NF-kB.2 Previous P100 proteolysis allows nuclear translocation of these complexes.7 results demonstrated that preserved NF-kBactivityand Most reports addressing the question of NF-kB activation by protection against apoptosis would be the minimal LMP1 used reporter gene assays or studied the involvement of prerequisite for all LMP1 natural mutated variants isolated NEMO/IKKa/IKKb or NF-kB-inducing kinase/IKKa, and most studies from both normal and Reed-Sternberg cells from Hodgkin’s were performed in non-B-cell lineages.8–11 Very few studies have lymphomas.3 addressed the question of the specific roles of RelA and RelB in 1CNRS-UMR-7276, University of Limoges, and CHU Dupuytren, Laboratory of Hematology, Limoges, France; 2CHU Dupuytren, Laboratory of Pathology, Limoges, France; 3CHU de Bordeaux, Laboratory of Pathology, Bordeaux, France and 4Department of Hematology, CHU Dupuytren, Limoges, France. Correspondence: Dr N Faumont, CNRS-UMR-7276, Hospital University Center (CHU) Dupuytren, University of Limoges, Laboratory of Hematology, 2 rue du Docteur Marcland, Limoges 87025, France. E-mail: [email protected] 5Present address: Statistical Unit, IFCS (Institut de formation aux carrie`res de sante´), Rabat, Morocco. Received 31 July 2013; revised 13 September 2013; accepted 17 September 2013; accepted article preview online 23 September 2013; advance online publication, 15 October 2013 NF-kB in EBV-transformed B cells A Chanut et al 872 EBV-transformed B cells. This question is of importance not only to RNeasy mini kit (Qiagen, Valencia, CA, USA). High quality (integrity and purity) understand the place of both subunits in B-cell transformation but of RNA was verified by the Agilent RNA 6000 Nano LabChip kit and the also in the view of developing new drugs targeting NF-kB. Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Here, we addressed the question of the function, DNA-binding activity and gene expression signatures of RelA and RelB in Gene expression profiling EBV-immortalized B-cell line models. Amplification of RNAs and hybridization onto microarrays were performed on an Affymetrix Gene Atlas system with the Affymetrix Human Genome U219 Array. Data analysis was performed according to both the LIMMA and MATERIALS AND METHODS SAM methods.21–23 Biological functions of genes were studied using ‘Gene Patients and biopsies Set Enrichment Analysis’ (www.broadinstitute.org/gsea/index.jsp). Details For this study, 11 patients, three with EBV-positive posttransplant DLBCLs are in Supplementary Materials and Methods. (EBV-DLBCL n1 1–3, following kidney transplantation for patients 1 and 2 and curative bone marrow transplantation for T-cell lymphoma for patient 3; Gene quantification with TaqMan low density array patient 1 was under mycophenolate and tacrolimus and patients 2 and 3 cDNAs were reverse transcribed from total RNA samples using the High were under cyclosporine A regimen. Patient 3 initially received one dose of Capacity cDNA Archive Kit (Life Technologies, Carlsbad, CA, USA). PCR antilymphocyte globulins), one with EBV positive human immuno- products were amplified from 200 ng of each cDNA sample using the 1 deficiency virus-associated DLBCL (EBV-DLBCL n 4), four with EBV-positive TaqMan Universal PCR Master Mix and TaqMan Gene Expression 1 DLBCLs of the elderly (EBV-DLBCL n 5–8), and three with nontumoral Assays preloaded in each reaction well of TaqMan low density arrays 1 reactive lymph nodes (LN n 1–3), were enrolled according to institutional (Life Technologies). Thereafter, TaqMan low density arrays were run on the regulations and after approval by the IRB of the university hospital of 7900HT system for quantitative real-time PCR analysis. Details are in Limoges. EBERs were detected in all tumor cells. Tumor cells expressed Supplementary Materials and Methods. CD20 and LMP1 confirmed by immunohistochemical analysis in all cases. Plasmid constructs RESULTS Complementary DNAs for LMP1,12 Luciferase (Promega, Paris, France), RelA and RelB differentially regulate proliferation and survival in 12 13 13 IkBaS32,36A, RelA, RelB and p100/p52 were cloned into the previously EBV-immortalized B cells 14 described pRT-1 doxycycline-inducible episomal vectors. The RelA insert To assess functional roles of RelA and RelB, we cloned the was obtained by long distance amplification of the corresponding mRNA from an LCL cell line. corresponding cDNAs and that of their respective inhibitor, IkBaS32,36A super-repressor and p100, into the doxycycline- regulatable pRT-1 vector.14 The same vector coding for Cell lines, CD40-Ligand stimulation and cell transfection Luciferase15 was used as control. After estradiol deprivation to Classical lymphoblastoid cell lines PRI, 1602, TSOC, LCL.4 and LCL.6 have 3,15 induce arrest of the EBV-latency III program, EREB2–5 B cells were been described in previous reports. EREB2–5 cells are a nonclassical LCL either pre-exposed or not to doxycyline for 24 h and then treated cell line with an estradiol-inducible EBV-latency III proliferation program with estradiol for both 24 and 48 h. In this model, estradiol due to an estrogen receptor fused to the EBNA2 viral protein.16 treatment of cells induces the EBV-latency III program with LMP1 Transfection, hygromycin selection and CD40-Ligand stimulation of 24 EREB2–5 cells were performed as previously described.17,18 expression that induces both NF-kB activation pathways. At 48 h, overexpression of each protein was checked by Luciferase assays or by western blot (Figure 1a).