Rela and Relb Cross-Talk and Function in Epstein–Barr Virus Transformed B Cells

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). As expected, Protein extracts and electrophoretic mobility shift assays (EMSAs) EBV EBV 19 induction of the EBV-latency III program (E24h and E48h ) Methods for cytoplasmic and nuclear extracts are described elsewhere. reverted spontaneous apoptosis and induced cell growth in The PRE double-stranded oligonucleotide probe and the EMSA technique estradiol-starved E0hEBV cells in Luciferase-expressing cells are described in Supplementary Materials and Methods. (Figures 1b and c). RelA induction by doxycycline weakly over- increased protection against apoptosis and markedly over- Immunoprecipitation and western blot increased cell proliferation when compared with Luciferase or Immunoprecipitation of p50 was performed as described in uninduced EBV-latency III proliferating control cells, respectively Supplementary Materials and Methods. Western blots were performed as 20 (Figures 1b and c). Induction of the super-repressor form of IkBa, previously described. The antibodies used are detailed in Supplementary IkBa , which inhibits the classical pathway,19,25 abolished Materials and Methods. S32,36A both EBV-induced protection against apoptosis and growth (Figures 1b and c). Surprisingly, induction of both RelB and p100 Analysis of apoptosis by flow cytometry decreased EBV-latency III protection against apoptosis (Figure 1b). Cells were double stained with AnnexinV-FITC (BD Pharmingen, San Diego, RelB also repressed EBV-induced proliferation of EREB2–5 cells, CA, USA) and propidium iodide (Sigma-Aldrich, Saint-Louis, MO, USA) in 20 whereas p100 had no effect (Figure 1c). These results clearly cold PBS-CaCl2-MgCl2 (Invitrogen, Cergy-Pontoise, France) as described suggest that RelA activation was associated with both proliferation and analyzed by FACS Calibur cytometer (BD Pharmingen, Paris, France). and protection against apoptosis in EBV-latency III immortalized B cells. The role of RelB on apoptosis and proliferation would be Proliferation assays more complex. Transfected EREB2–5 cells were plated in 96-well plates (103/well), in 10% fetal calf serum medium containing or not doxycycline. Over 2 days, Dynamics of NF-kB complexes in EBV-immortalized B cells: proliferation rates were measured using the CellTiter 96 AQueous One reciprocal inhibition of RelA and RelB Solution Cell Proliferation Assay (Metabolism Tetrazolium Salt assay from Promega). To correlate functional assays with NF-kB DNA binding activities, we performed EMSA. Controls for specificity are given in Cell sorting and RNA isolation Supplementary Figure S1. First, nuclear extracts from five EBV-latency III immortalized B cell lines, all with LMP1 expression, EREB2–5 cell line conditions used for gene expression profiling are detailed in Supplementary Materials and Methods. When indicated, NGFRt-expressing were isolated. NF-kB DNA binding patterns systematically EREB2–5 transfected cells were purified with MACS microbeads corresponded to three bands, BI, BII and BIII (Figure 2a). (Miltenyi Biotec, Bergisch Gladbach, Germany) following previously published Supershift experiments indicated that band B-I was likely to protocols.12 Isolation of the high-quality total RNA was performed from correspond to RelA/p50 dimers, band B-II to both c-Rel and RelB samples (cell lines and tissues) in the TRIzol Reagent (Invitrogen) using the complexes and band B-III to p50 homodimers. Kinetics of

Leukemia (2014) 871 – 879 & 2014 Macmillan Publishers Limited NF-kB in EBV-transformed B cells A Chanut et al 873

Figure 1. Effect of RelA, IkBaS32,36A, RelB and p100 on apoptosis and proliferation of EBV-latency III program-induced EREB2–5 cells. EREB2–5 cells were stably transfected with the doxycycline-regulatable pRT-1 vector coding for Luciferase, RelA, IkBaS32,36A, RelB or p100. After estradiol starvation for 72 h, cells were treated ( þ ) or not ( À ) with doxycycline (Dox.) for 24 h. Then, the EBV-latency III program was re-induced or not (E0hEBV) by addition of estradiol for 24 (E24hEBV) or 48 h (E48hEBV). (a) At 48 h, analysis of Luciferase activity (lanes 1–2) and western blots for the expression of RelA (lanes 3–4), IkBaS32,36A (lanes 5–6), RelB (lanes 7–8) p100/p52 (lanes 9–10) and a-tubulin (aTub., lanes 3–10). (b) Annexin-V labeling was assessed by flow cytometry on estradiol-starved doxycycline-treated pRT-1-transfected EREB2–5 cells (E0hEBV) and after 24 (E24hEBV) or 48 h (E48hEBV) estradiol re-induction of the EBV-latency III program in the presence of doxycycline. For each pRT-1-transfected EREB2–5 cells, percentages of Annexin-V-positive cells were normalized to those of E0hEBV cells, corresponding to 100%. Statistically significant differences (one-way ANOVA) between the three time points E0hEBV, E24hEBV and E48hEBV are indicated as follows: *(Po0.05), **(Po0.01), ***(Po0.001) and ****(Po0.0001). ns, non-significant. (c) Proliferation rates were assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Metabolism Tetrazolium Salt assay from Promega). Relative proliferation rate was defined as the ratio of the absorbance at 492 nm between estradiol-starved pRT-1-transfected EREB2–5 cells (E0hEBV) and 24 (E24hEBV) or 48 h (E48hEBV) estradiol re-induction of the EBV-latency III program in the presence or not of doxycycline. T-test significant differences between cells treated or not with doxycycline at the same time point are indicated by *(Po0.05) and **(Po0.01). Each experiment was performed at least three times.

EBV-latency III program induction in EREB2–5 cells showed that completely supershifted with c-Rel antibody (Figure 3b, lanes NF-kB activation paralleled LMP1 expression levels and reached 2, 3 and 8). Thus, RelA overexpression repressed RelB but not maximum rates at 6 h (Figure 2b, lanes 1–5). After LMP1 c-Rel DNA binding activity. The expression of IkBaS32,36A led to a expression or stimulation of its cell homolog CD40 for 24 h, the global decrease in NF-kB DNA binding activity (Figure 3c, lane 2). same NF-kB complexes with the same bands BI, BII and BIII were Over-expression of RelB deeply modified the NF-kBDNA identified (Figure 2b, lanes 7–18). At the protein level, NF-kB binding activity, with two major bands: one corresponding to subunits of the classical pathway, that is, RelA- and c-Rel- B-II and one new band, called B-IV (Figure 3d, lanes 2 and 3). Both containing complexes, were rapidly translocated into the nucleus bands B-II and B-IV were completely supershifted with RelB starting at 1 h after EBV-latency III induction (E1hEBV), whereas antibodies (Figure 3d, lane 6). As attested by the absence of RelB complexes were activated later at 24 h (E24hEBV,Figure2c). supershift with RelA and c-Rel antibodies, RelB overexpression These results clearly show a rapid and strong activation of the repressed NF-kB complexes activated by the classical pathway classical NF-kB pathway and much delayed, slower and weaker (Figure 3d, lanes 4 and 8). Moreover, band B-IV was completely activation of the alternative pathway by the EBV-latency III supershifted with p50 antibodies, suggesting that p50 was the program. main partner of RelB (Figure 3d, lane 5). Finally, overexpression of Second, we analyzed NF-kB complexes in EREB2–5 cells p100 did not affect RelA DNA binding activity and specifically transfected with RelA, RelB, IkBaS32,36A super-repressor and induced the loss of RelB binding activity in band B-II, such a loss p100 after 48 h EBV-latency III program induction. Luciferase wassuggestedbythecompleteextinctionofbandB-IIinthe expression had no effect on NF-kB binding activity (Figure 3a, presence of c-Rel antibody (Figure 3e, lane 8). This result can be lane 2). RelA induction was associated with (i) an increase in interpreted as a specific inhibition of RelB DNA binding activity band B-I containing the RelA/p50 and RelA/RelA dimers by an excess of p100 protein, which would trap RelB in the (Figure 3b, lanes 2–5) and (ii) a decrease in band B-II, now cytoplasm.

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Figure 2. Activation of NF-kB pathways in EBV-latency III immortalized B cells. (a) NF-kB DNA binding activity from nuclear extracts of ﬁve classical EBV-latency III immortalized lymphoblastoid B cell lines. Lane 1 to 5: EMSA with the PRE radiolabeled probe for LCL PRI (lane 1), 1602 (lane 2), TSOC (lane 3), LCL.4 (lane 4) and LCL.6 (lane 5). Corresponding western blots for LMP1 and aTub. are shown below. Lane 6–11: supershift experiments from TSOC nuclear extracts using antibodies against Oct2 (irrelevant control, lane 6), RelA (lane 7), p50 (lane 8), RelB (lane 9), p52 (lane 10) and c-Rel (lane 11). (b and c) Kinetic experiments of NF-kB activation on EREB2–5 cells. Estradiol-starved cells were treated for 0, 1, 3, 6 and 24 h with estradiols E0hEBV, E1hEBV, E3hEBV, E6hEBV and E24hEBV, respectively. Estradiol-starved cells were also treated for 24 h with CD40 Ligand (E24hCD40). (b) Lanes 1–6: EMSA for NF-kB DNA binding activity of nuclear extracts from E0hEBV to E24hEBV and E24hCD40 cells (upper panel) with corresponding western blots for LMP1 (middle panel) and aTub.(lower panel). Lanes 7–18: supershift assay of nuclear extracts from E24hEBV (lanes 7–12) and E24hCD40 cells (lanes 13–18). (c) Lanes 1–6: kinetics of nuclear translocation of NF-kB subunits. Western blots from nuclear and cytosolic extracts for p50, RelA, c-Rel, p52 and RelB proteins. Revelations of aTub. and SAM68 were used for loading controls of cytosolic and nuclear extracts, respectively. For EMSAs, bands B-I to B-III are indicated by arrows and supershifted bands are indicated by * for each lane.

Mechanisms of RelA and RelB reciprocal inhibition inhibit binding of all RelB/p50 heterodimers to DNA, as attested by As p50 was the main partner of RelA and RelB by EMSA, we looked the EMSA experiments presented in Figure 3. for the p50 partners by immunoprecipitation. P50 was indeed Altogether, results presented in Figure 3 and 4 demonstrated a associated with RelA, RelB and p52 in Luciferase control cells cross-talk between RelA and RelB so that RelA was likely to inhibit (Figure 4, lane1). Induction of RelA was associated with a marked RelB DNA binding by overexpression of p100 and RelB repressed increased in immunoprecipitated RelA/p50 heterodimers and a RelA DNA binding by competing for their association with p50. slight increase in immunoprecipitated RelB/p50 complexes (Figure 4, lane 2). Furthermore, p50 was additionally associated with p100 in these RelA-overexpressing cells, which correlates RelA and RelB transciptomic signatures with an increase in p100 expression in the input (Figure 4, lanes 1 To evidence cross-talk between RelA and RelB at the transcrip- and 5). These results led to the hypothesis that RelA over- tional level, that is, their cross-inhibition, we searched traces of expression could indirectly repress DNA binding of RelB/p50 such interplay using high-throughput gene expression profiling. complexes by increasing p100 expression, which in turn would We first established the list of genes deregulated in EBV- trap RelB-containing complexes in the cytoplasm. transformed B cells. To avoid gene deregulation due to in vitro cell When RelB was overexpressed, RelB/p50 dimers were strongly line artifacts, we also addressed genes truly deregulated in EBV- increased and, concomitantly, RelA/p50 complexes were associated DLBCLs with the expression of LMP1 (three cases of decreased when compared with Luciferase control cells EBV-related posttransplant DLBCL, one case with EBV-positive (Figure 4, lane 3). In this condition, p100 expression and human immunodeficiency virus-associated DLBCL and four with association with p50 were also increased (Figure 4, lanes 3 and 6). EBV-positive DLBCLs of the elderly). Such variants of EBV- This p100 induction by RelB was likely to exert some inhibitory associated DLBCLs would also allow to prevent any bias due to negative feedback on RelB but was certainly not sufficient to a particular subtype of EBV-associated B-cell lymphomas.

Leukemia (2014) 871 – 879 & 2014 Macmillan Publishers Limited NF-kB in EBV-transformed B cells A Chanut et al 875

Figure 3. NF-kB DNA binding activity after RelA, IkBaS32,36A, RelB or p100 induction. EREB2–5 cells were stably transfected with the doxycycline-regulatable pRT-1 vector coding for Luciferase, RelA, IkBaS32,36A, RelB and p100. After estradiol starvation, cells were treated ( þ ) or not ( À ) with doxycycline (Dox.) for 24 h. Then, the EBV-latency III program was re-induced by addition of estradiol for 48 h. (a to e, lanes 1 and 2) EMSA for NF-kB DNA binding activity of EREB2–5 cells transfected with (a) pRT-1-Luciferase vector, (b) pRT-1-RelA, (c) pRT-1-IkBaS32,36A, (d) pRT-1-RelB and (e) pRT-1-p100. (b, d and e, lanes 3–8) The supershift assay of nuclear extracts from cells treated with both estradiol and doxycycline. Nuclear extracts were incubated with anti-Oct2 (irrelevant control, lane 3), anti-RelA (lane 4), anti-p50 (lane 5), anti-RelB (lane 6), anti-p52 (lane 7) or anti-c-Rel (lane 8) antibodies. Bands B-I to B-IV are indicated by arrows, and supershifted bands are indicated by * for each lane.

On one hand, the different B cell lines were divided in two groups, those in which the EBV-latency III program was on or off. Cells expressing LMP1 alone were also included (Supplementary Figure S2). Selected genes yielded a cell line list of 1550 genes upregulated by the EBV-latency III program (Supplementary Table S1). On the other hand, EBV-DLBCL tumors were compared with three non-tumoral lymph nodes with benign follicular hyperplasia. This led to an EBV-related tumor list of 3415 genes (Supplementary Table S2). A total of 726 genes were in common between both lists, being thus bona fide EBV-induced genes in EBV-transformed B cells both in vivo and in vitro (Supplementary Figure S3 and Supplementary Table S3). Among these 726 genes, 612 (84%) were regulated by LMP1 alone and numerous genes were known targets of c-Myc, E2F1, Jun or NF-kB (Supplementary Figure S3 and Supplementary Table S3). Then, EREB2–5 cells overexpressing Luciferase, RelA, RelB, IkBaS32,36A super-repressor and p100 were sorted after doxycycline induction (i.e., E.Luc, E.RelA, E.RelB, E.IkBaSS32,36A and E.p100 cells, respectively), and their gene expression profiles were analyzed to identify RelA and RelB target genes among the 726 EBV upregulated genes. Forty-six genes were very likely to be upregulated by RelA and/or RelB in EBV-DLBCL-transformed B cells (Supplementary Materials and Methods for details and Figure 5). Of note, 44/46 Figure 4. p50 Immunopreicipitation after induction of RelA and genes were upregulated by LMP1 with a fold change ranging from RelB. EREB2–5 cells were stably transfected with the doxycycline- 1.6–85 (Supplementary Table S4). As an independent experiment, regulatable pRT-1 vector coding for Luciferase, RelA or RelB. After we looked at the deregulation of these genes in the series estradiol starvation, cells were treated with doxycycline for 24 h and published by Basso et al.26 (GEO Accession Number: GSE2350), then the EBV-latency III program was re-induced by addition of estradiol for 48 h. Western blot detection of RelA, p105/p50, RelB which includes purified resting B cells from cord blood, and p100/p52 from immunoprecipitated p50 and input extracts of lymphoblastoid cell lines and immunoblastic DLBCLs from both EREB2–5 overexpressing Luciferase (lanes 1 and 4), RelA (lanes 2 AIDS-related immunodeficient and immunocompetent patients. and 5) and RelB (lanes 3 and 6). Input control by western blot for Indeed, these DLBCL variants are frequently associated with aTub. are shown (lanes 4–6). constitutive NF-kB activation.27,28 Of the 46 RelA and/or RelB

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Figure 5. RelA and RelB traces among EBV-latency III-deregulated genes in EBV-transformed B cells. The different cell line conditions are EBV EBV EBV EBV indicated at the top of the panels: E0h À 120h þ Dox-Luc, Econt þ Dox-Luc, Econt þ Dox-RelA, Econt þ Dox-IkBaS32,36A, EcontEBV þ Dox-RelB and EcontEBV þ Dox-p100. Genes were arrayed in a descending order according to their expression level in EcontEBV þ Dox-Luc. Gene symbols are indicated on the right. RelA- or RelB-regulated genes were those that were overexpressed in E.RelA or E.RelB when compared with E.IkBaS32,36 A or E.p100 cells, respectively, and that were downregulated in E.IkBaSS32,36A or E.p100 cells, respectively when compared with E.Luc cells. Expression proﬁles of (a) 11 RelA and RelB co-dominant target genes, corresponding to intersection between the RelA and RelB lists, (b) 24 RelA dominant target genes, corresponding to the RelA but no to the RelB list, and (c)11 RelA or RelB target genes only, corresponding to genes whose expression was repressed by RelB or RelA, respectively. (d) List of RelA (n ¼ 45) and RelB target genes (n ¼ 12) among the 726 upregulated genes by EBV in LCLs and EBV-DLBCLs. Log2-value color codes are shown below each panel. EREB2–5 line conditions are detailed in Supplementary Materials and Methods. Note the induction of NFKBIA/IKB mRNA in the EBV EBVcont þ Dox-IkBaS32,36A condition (IkBaS32,36A overexpression).

target genes, 34 could be retrieved from the gene list of Basso genes were also induced by RelB, that is, RelA- and RelB-co- et al., and among them 31 were indeed deregulated either in regulated genes (Figure 5a). Twenty four of these 45 genes were lymphoblastoid cell lines or in immunoblastic DLBCLs apparently not regulated by RelB (so-called RelA-dominant genes) (Supplementary Table S5). Deregulation of these genes was (Figure 5b). This can be interpreted as a simultaneous and conﬁrmed by quantitative PCR on cDNA from EBV-DLBCL tumors overlapping RelA repression by RelB and RelB weak gene and LCL cells (Supplementary Figures S4 and S5). upregulation. The remaining 11 RelA-regulated genes were Among the RelA and/or RelB target genes selected by our repressed by RelB, so-called RelA-only genes (Figure 5c). RelB analysis, 45/46 were induced by RelA (Figure 5). Eleven of these 45 induced the expression of one gene, ARNTL2 (Aryl hydrocarbon

Leukemia (2014) 871 – 879 & 2014 Macmillan Publishers Limited NF-kB in EBV-transformed B cells A Chanut et al 877 receptor nuclear translocator-like protein 2), whose expression b receptor stimulation.41 Consistently, as the overexpression of RelA was repressed by RelA (Figure 5c). increased p100 and RelB expression and RelB/p50 association These results clearly suggest that RelB may repress transcription together with decreased RelB DNA binding activity, we interpreted of some RelA target genes and vice versa. Additionally, it appeared these results as the effect of RelB trapping by p100. However, that RelB signature was almost entirely included in the one of RelA another alternative would be that RelA and RelB association would (Figure 5d). directly inhibit RelB DNA binding activity, as reported in the Functions of RelA- and/or RelB-regulated genes are shown in murine embryonic fibroblast model.42 In any case, DNA binding of Supplementary Figure S6 and Supplementary Table S6. Genes RelB was always weak in EBV-latency III immortalized B cells when dominantly regulated by RelA were associated with metabolism, compared with that of RelA or c-Rel or with RelB activity induced genetic instability, protection against apoptosis, cell cycle and by CD40 stimulation. Kinetic experiments on induction of the immune response. Additional functions of genes codominantly EBV-latency III program demonstrated that RelA complexes were regulated by RelA and RelB were autophagy, transcription factors primarily translocated into the nucleus and paralleled LMP1 regulating cell cycle and plasma cell differentiation and NF-kB expression. Upon EBV stimulation, RelB activation was observed regulators. The only RelB-specific gene (i.e., repressed by RelA) was later at 24 h and was concomitant with TRAF3 degradation and ARNTL2, a gene involved in the regulation of circadian rhythm and p100 cleavage to p52 (not shown). Thus, not only RelB is protection against hypoxia. secondarily induced by RelA but also it is negatively retro- controlled by RelA. To further search for functional traces of RelA and RelB DISCUSSION interactions in EBV-related DLBCLs, we established gene expres- NF-kB pathways have been implicated in Hodgkin’s lymphomas, sion profiles of both our B-cell line models and EBV-associated multiple myelomas and both EBV-negative and EBV-associated DLBCL tumors. A total of 726 six genes were upregulated in both DLBCLs.29–33 Recently, it has been demonstrated in a mouse tumors and cell lines, most of them being regulated by the LMP1 transgenic model that LMP1-expressing B cells are under oncogene of EBV. On the basis of RelA and RelB inhibition or permanent control of both T-cell-acquired and NK cell innate activation experiments, 46 genes were found to be regulated immune responses.34 We indeed demonstrated that LMP1 is able either by RelA or RelB. The number of RelA- and/or RelB-regulated to promote autologous T cell targeting of EBV-infected B cells in genes seemed to be low, when compared with the number of humans.12 Despite this permanent immune control, LMP1 remains genes induced by LMP1 alone, and probably not all NF-kB target the main oncogenic agent of EBV, as demonstrated both in genes were selected following our methodology. However, LMP1 human EBV-related tumors and different mice models, including is a very strong activator, and overexpression of IkBaS32,36A or the model developed by Zhang et al.34 Indeed, almost all p100 could have not completely abolished NF-kB DNA binding LMP1-positive EBV-associated B-cell lymphomas are associated activity in EBV-immortalized B cells. Moreover, as tissue control for with a more or less profound immunodeficiency status. The EBV-related DLBCLs, we took lymph nodes with benign follicular oncogenic potential of EBV is directly linked to continuous activation hyperplasia, that is, lymph nodes with current intense secondary of NF-kBbyLMP1.35 We thus worked on LMP1-expressing EBV- B-cell immune response. Thus, it is not excluded that some NF-kB transformed B cells, raising the question of the respective roles of activation would exist in these control tissues. We can hypothesize RelA and RelB. The model that emerged from our results is that RelA that we detected the most NF-kB-sensitive genes deregulated in and RelB cross-talked together so that RelB was subordinated to DLBCLs when compared with benign lymph nodes submitted to RelA and would help RelA in reinforcing the regulation of some an intense immune response. genes like those involved in autophagy or metabolism. The expression of ARNTL2 gene was induced by RelB and As a side conclusion of our work, we found that p50 was the repressed by RelA overactivation (so-called RelB only). Reciprocally, main partner of RelA and RelB in EBV-immortalized B cells. 10 RelA-regulated genes were repressed by RelB overexpression Overexpressed RelB trapped most, if not all, available p50 subunits (so-called RelA only). These results are a strong indication that in the cell, inhibiting therefore DNA binding of both RelA/p50 and RelA and RelB reciprocal inhibition may occur at the transcrip- c-Rel/p50 complexes. Thus, in EBV-infected B cells, RelA and RelB tional level in transformed B cells. Genome-wide gene expression were in competition for p50, explaining mechanistically how RelB analysis derived from Lymphotoxin b receptor-stimulated murine exerted an inhibitory activity on RelA. Although poorly repre- embryonic fibroblasts revealed that the majority of the induced sented in our cell models, RelB/p52 complexes were also increased genes require both RelA- and RelB-containing dimers.43 Here, RelB under RelB overexpression. Although processing of p105 to p50 is gene expression profile was almost included in that of RelA. As constitutive, p100 degradation or processing to p52 results from RelB activity was secondarily induced by RelA in EBV-transformed the activation of alternative signaling, allowing nuclear transloca- B cells and genes repressed by RelB in our experimental in vitro tion of RelB-containing dimers associated with p50 or p52, conditions were in fact overexpressed both in vitro and in respectively.36 The nfkb2/p100 À / À -deficient mice model showed EBV-related DLBCLs, it is very likely that the optimal equilibrium a functional overlap between p50 and p52 when bound to RelB.37 between RelA and RelB would exist in EBV-transformed B cells so As RelB blocks RelA DNA binding by trapping its main partner, the that RelA-specific functions remained and RelA/RelB co-regulated p50 subunit, it can be put forward that the inhibitory effect of RelB functions were secondarily reinforced. overexpression on both proliferation arrest and increased apoptosis Taking both RelB and RelA together, one of the main NF-kB corresponds to its inhibitory effect on RelA. It is only because p100 signatures was that of metabolism with regulation of genes overexpression was associated with both increased apoptosis and involved, for example, in cholesterol synthesis, fatty acid specificlackofRelBDNAbindingactivitythatwecaninferthatRelB degradation or the pentose phosphate pathway. Recent publica- is likely to have a role in protection against apoptosis. tions have identified NF-kB as a major regulator of energy Several recent studies provided data that interconnections homeostasis via the regulation of metabolic functions.44 Here, between the alternative and classical NF-kB pathways exist ACSL1 (acyl-CoA synthetase long chain family, member 1) codes through expression control of NF-kB subunits.38 Regulatory for an enzyme that produces acetyl-CoA, the requisite building regions of relb and nfkb2/p100 genes contain kB binding sites, block for lipid biosynthesis, and also contributes to energy and the expression of these genes is dependent on RelA.39,40 In production through the b-oxidation into mitochondria.45,46 addition, experiments on rela À / À murine embryonic fibroblasts Tumor cells reactivate de novo lipid synthesis such as that of suggested that RelA-dependent expression of RelB is the main fatty acids, which are important substrates for energy production, determinant of alternative pathway responses through Lymphotoxin essential components of all biological lipid membranes and

& 2014 Macmillan Publishers Limited Leukemia (2014) 871 – 879 NF-kB in EBV-transformed B cells A Chanut et al 878 participate in several signaling pathways that regulate in turn Re´gional du Limousin, Comite´s Limousin de la Ligue contre le Cancer and Association metabolism, proliferation, survival and invasive properties.47 pour la Recherche sur le Cancer. We thank the tumor banks of the University Consistently with functional results, the RelA signature indicated hospitals of Bordeaux and Limoges for providing biopsies and Ve´ronique Pantesco that it is very likely to participate in strong proliferation of EBV- (Microarray Core Facility of the Institute for Research in Biotherapy, Montpellier, transformed B cells. Indeed, other RelA-regulated genes revealed France) for Affymetrix analyses. We thank Dr Jeanne Cook-Moreau, CNRS-UMR-7276 Limoges, for English corrections and Mr Lionel Forestier platform GENOLIM, that RelA was in the center of key functions associated with cell University of Limoges, France. N Faumont was supported by Comite´ Orientation transformation, including regulation of genes involved in cell cycle Recherche Cancer du Limousin, A Chanut and S Durand-Panteix by Association pour progression such as BATF (basic leucine zipper transcription factor, la Recherche sur le Cancer. A Chanut was also supported by Socie´te´ Française ATF-like) and CCNE1 (cyclin E1). The B-cell-specific transcription d’He´matologie. factor, BATF, promotes latent EBV infection with transforming potential and contributes to in vivo progression of EBV-associated lymphomagenesis.48–50 G1/S checkpoint alteration by cyclin E1 REFERENCES (CCNE1) overexpression is associated with high cell proliferation 1 Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. 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