EBV Gene Expression Research Articles

Methionine restriction forces Epstein-Barr virus out of latency

EBV gene expression is repressed during viral latency to prevent an immune response, but it is not known how metabolism contributes to this silencing. In this issue of Cell Metabolism, Guo etal. describe how methionine restriction reactivates the expression of EBV genes, offering new therapeutic approaches against EBV-driven diseases.

Abstract 774: Microdissected gene expression profiling of recurrent nasopharyngeal carcinoma

Abstract Nasopharyngeal cancer (NPC) is an EBV-driven epithelial carcinoma with a recurrence rate of 30%. Biological pathways and predictive markers for recurrence remain understudied. Further, NPC tumors are associated with an intense inflammatory infiltrate, masking gene expression signals from the tumor epithelial compartment. We perform laser-capture microdissection on a preliminary cohort of 12 tumors, including primary tumors from recurrent and non-recurrent cases matched for stage of disease. Smart-3SEQ, a novel 3’ end RNA-Seq technique which allows for the accurate quantification of transcript abundance in FFPE samples, was performed to obtain gene expression libraries. Tumor epithelial and microenvironment regions from were separately microdissected, giving a total of 42 histologically-pure gene expression libraries. Raw reads were mapped to the human hg19 and EBV genomes and gene expression counts were obtained with Subread. Differential gene expression and gene set enrichment was performed in R with DESeq2 and fgsea respectively. We observed that primary NPC tumors that did not recur were significantly enriched for immune processes, including leukocyte mediated immunity, innate immune response and lymphocyte activation (padj < 1E-9 for all). The top differentially expressed genes in this group included mediators of inflammation such as STAT1 (padj = 0.0204). Critically, these gene expression changes were observed within the tumor epithelial compartment. In contrast, primary NPC tumors that eventually recurred were enriched for known oncogenes including BCL2 (padj = 0.000420). These findings suggest that heterogeneity in gene expression between NPC tumors play an important role in treatment response. Further work to define gene expression subtypes of NPC tumors will be helpful for patient stratification and personalized care. Citation Format: Joshua Tay, Wei Keat Teo, Chuan Keng Goh, Bing Cheng Wu, Kwok Seng Loh. Microdissected gene expression profiling of recurrent nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 774.

M6A Regulators Mediated Methylation Modification Patterns and Tumor Microenvironment Infiltration Characterization In Nasopharyngeal Carcinoma.

BackgroundThe role of RNA N6-methyladenosine (m6A) modification in tumor progression and metastasis has been demonstrated. Nonetheless, potential biological function of m6A modification patterns in nasopharyngeal carcinoma (NPC) remains unknown.MethodsThe m6A modification patterns were comprehensively evaluated based on 26 m6A regulators in NPC, and m6A subtype and also m6A score were identified and systematically correlated with representative tumor characteristics.ResultsTwo distinct m6A subtypes were determined and were highly consistent with immune activated and immune suppressed phenotypes, respectively. More representative m6A scores of individual tumors could predict tumor microenvironment (TME) infiltration, mRNA based stemness index (mRNAsi), EBV gene expression, genetic variation, and prognosis of NPC patients. Low m6A score, characterized by activation of immunity and suppression of mRNAsi and EBV gene, indicated an activated TME phenotype and better PFS and also lower risk of recurrence and metastasis. High m6A score, characterized by activation of Wnt and NF-κB signaling pathway and lack of effective immune infiltration, indicated an immune suppressed TME phenotype and poorer survival. Low m6A score was also correlated with increased tumor mutation burden (TMB) and better response to immunotherapy, and vice versa. A significant therapeutic advantage in patients with low m6A score was confirmed with an anti-PDL1 immunotherapy cohort.Conclusionsm6A patterns played an important role in the diversity and complexity of TME. m6A score could be used to evaluate the m6A pattern of individual tumor to enhance our understanding of TME infiltration and guide more effective immunotherapy strategies.

HHV-6 in the Lymphoma Microenvironment: Both Chicken and Egg?

HHV-6 in the Lymphoma Microenvironment: Both Chicken and Egg?

Overall Status of Epstein-Barr virus Infection, IFN-a, and TLR-7/9 in Patients with Systemic Lupus Erythematous.

Systemic lupus erythematous (SLE) is a multisystem autoimmune disorder. While studying the pathogenesis of SLE is prevalent, both infectious and non-infectious elements are regarded to exert an important impact on the disease's development. To explore the overall status of EBV, TLR7, TLR9, and IFN-α gene expression in 32 patients suffering from SLE and 32 healthy controls. Plasma and PBMCs were separated from fresh whole blood. To measure EBV DNA load and mRNA levels of IFN-a, TLR-7 and9 in PBMCs, molecular techniques were employed. The production of IFN-α, ds-DNA IgG antibody, and EBNA-1 IgG levels were also measured in plasma by ELISA. SLE patients showed significantly higher EBV load (p=0.001) and transcriptional levels of TLR7 (p=0.0001), IFN-α (p=0.0001), and TLR9 (p=0.0001) than controls. Moreover, the plasma levels of IFN-α (p=0.0002) and EBNA-1specific IgG antibodies (p=0.01) were significantly higher in SLE patients. The results stressed on the potential role of EBV infection and TLRs in SLE patients although more research is needed to determine the global impact that EBV infection can have on immune signature in patients with SLE.

Epstein-Barr Virus Episome Physically Interacts with Active Regions of the Host Genome in Lymphoblastoid Cells.

The Epstein-Barr virus (EBV) episome is known to interact with the three-dimensional structure of the human genome in infected cells. However, the exact locations of these interactions and their potential functional consequences remain unclear. Recently, high-resolution chromatin conformation capture (Hi-C) assays in lymphoblastoid cells have become available, enabling us to precisely map the contacts between the EBV episome(s) and the human host genome. Using available Hi-C data at a 10-kb resolution, we have identified 15,000 reproducible contacts between EBV episome(s) and the human genome. These contacts are highly enriched in chromatin regions denoted by typical or super enhancers and active markers, including histone H3K27ac and H3K4me1. Additionally, these contacts are highly enriched at loci bound by host transcription factors that regulate B cell growth (e.g., IKZF1 and RUNX3), factors that enhance cell proliferation (e.g., HDGF), or factors that promote viral replication (e.g., NBS1 and NFIC). EBV contacts show nearly 2-fold enrichment in host regions bound by EBV nuclear antigen 2 (EBNA2) and EBNA3 transcription factors. Circular chromosome conformation capture followed by sequencing (4C-seq) using the EBV origin of plasmid replication (oriP) as a "bait" in lymphoblastoid cells further confirmed contacts with active chromatin regions. Collectively, our analysis supports interactions between EBV episome(s) and active regions of the human genome in lymphoblastoid cells.IMPORTANCE EBV is associated with ∼200,000 cancers each year. In vitro, EBV can transform primary human B lymphocytes into immortalized cell lines. EBV-encoded proteins, along with noncoding RNAs and microRNAs, hijack cellular proteins and pathways to control cell growth. EBV nuclear proteins usurp normal transcriptional programs to activate the expression of key oncogenes, including MYC, to provide a proliferation signal. EBV nuclear antigens also repress CDKN2A to suppress senescence. EBV membrane protein activates NF-κB to provide survival signals. EBV genomes are maintained by EBNA1, which tethers EBV episomes to the host chromosomes during mitosis. However, little is known about where EBV episomes are located in interphase cells. In interphase cells, EBV promoters drive the expression of latency genes, while oriP functions as an enhancer for these promoters. In this study, integrative analyses of published lymphoblastoid cell line (LCL) Hi-C data and our 4C-seq experiments position EBV episomes to host genomes with active epigenetic marks. These contact points were significantly enriched for super enhancers. The close proximity of EBV episomes and the super enhancers that are enriched for transcription cofactors or mediators in lymphoblasts may benefit EBV gene expression, suggesting a novel mechanism of transcriptional activation.

A Genome-Wide Epstein-Barr Virus Polyadenylation Map and Its Antisense RNA to EBNA.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen associated with Burkitt's lymphoma and nasopharyngeal carcinoma. Although the EBV genome harbors more than a hundred genes, a full transcription map with EBV polyadenylation profiles remains unknown. To elucidate the 3' ends of all EBV transcripts genome-wide, we performed the first comprehensive analysis of viral polyadenylation sites (pA sites) using our previously reported polyadenylation sequencing (PA-seq) technology. We identified that EBV utilizes a total of 62 pA sites in JSC-1, 60 in Raji, and 53 in Akata cells for the expression of EBV genes from both plus and minus DNA strands; 42 of these pA sites are commonly used in all three cell lines. The majority of identified pA sites were mapped to the intergenic regions downstream of previously annotated EBV open reading frames (ORFs) and viral promoters. pA sites lacking an association with any known EBV genes were also identified, mostly for the minus DNA strand within the EBNA locus, a major locus responsible for maintenance of viral latency and cell transformation. The expression of these novel antisense transcripts to EBNA were verified by 3' rapid amplification of cDNA ends (RACE) and Northern blot analyses in several EBV-positive (EBV+) cell lines. In contrast to EBNA RNA expressed during latency, expression of EBNA-antisense transcripts, which is restricted in latent cells, can be significantly induced by viral lytic infection, suggesting potential regulation of viral gene expression by EBNA-antisense transcription during lytic EBV infection. Our data provide the first evidence that EBV has an unrecognized mechanism that regulates EBV reactivation from latency.IMPORTANCE Epstein-Barr virus represents an important human pathogen with an etiological role in the development of several cancers. By elucidation of a genome-wide polyadenylation landscape of EBV in JSC-1, Raji, and Akata cells, we have redefined the EBV transcriptome and mapped individual polymerase II (Pol II) transcripts of viral genes to each one of the mapped pA sites at single-nucleotide resolution as well as the depth of expression. By unveiling a new class of viral lytic RNA transcripts antisense to latent EBNAs, we provide a novel mechanism of how EBV might control the expression of viral latent genes and lytic infection. Thus, this report takes another step closer to understanding EBV gene structure and expression and paves a new path for antiviral approaches.

Abstract 3087: Identification of EBV infection and its life cycle in pan-cancer samples

Abstract Viral infection is associated with 10-15 percent of human cancers and EBV (human herpesvirus 4 (HHV-4)) contributes to cancer development in diverse cancer types. The life cycle of EBV is divided into latent and lytic cycles. Lytic replication cycle begins when early transcription factors are activated, and initiation complex is formed. Subsequently, lytic antigens are expressed once infected cells enter the lytic cycle. But the contribution of each cycle in cancer development is not well known yet. Hence we explored EBV integration and gene expression pattern by analyzing TCGA whole transcriptome sequencing (WTS) data.We investigated the integration of EBV by using FASTQ-formatted TCGA tumor WTS data. We designed a three-step EBV detection workflow. 1) Overall viral integration into human genome sequence was detected by VirusSeq. 2) “Spliced Transcripts Alignment to a Reference” algorithm was used for mapping against a hybrid reference genome of human and EBV. 3) To quantify the expression of EBV-related genes, we applied “RNA-Seq by Expectation Maximization” algorithm to obtain Transcripts Per Million values. Furthermore, we validated this detection workflow in EBV-positive cell lines from the public data repository and analyzed mutation profiles by using Mutation Annotation Format files.We investigated 851 WTS samples over 23 cancer types and scanned viral presentations by mapping against four types of EBV strains (Human HHV-4 complete wild-type, GD1, AG876, and artificial join). Consequently, we detected 88 samples mapped against the hybrid genome, and identified 46 of them were EBV-infected. By performing gene expression analysis, we recognized that 39/46 samples were expressing EBV lytic genes. Three types of cancers (Colorectal adenocarcinoma (COAD), Rectum adenocarcinoma (READ), and Stomach adenocarcinoma (STAD)) showed EBV lytic gene expression. Along with STAD, both BZLF1 and BALF2 were highly expressed in COAD; BZLF1 encodes an early transcription factor, and BALF2 is one of the viral genes constructs the initiation complex. Using our method, we also identified the expression of EBV lytic and latent genes in EBV-positive cell lines, hence validating our results. In addition, we found out an association between mutational events and EBV infection in COAD by profiling mutational signatures: increased C to T transitions and TP53 disruptions in EBV-infected samples. Overall, we could identify biologically relevant EBV-infected cancer samples considering EBV life cycle. We hereby drew two major conclusions based our findings. First, we established an analysis workflow to find out biologically meaningful EBV infection in cancer by quantifying gene expression of composing the EBV life cycle, beyond a simple viral integration. Second, we discovered EBV expression pattern in small number of colon cancer patients. Our integrative data will provide clues for comprehensively understanding of EBV-associated cancers. Citation Format: Hyojin Song, Hogune Im, Sung-Soo Yoon, Youngil Koh. Identification of EBV infection and its life cycle in pan-cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3087.

Abstract 4377: EBV associated gastric cancer in Korean

Abstract Gastric cancer was the third leading cause of death worldwide and most prevalent malignant cancer in Korea, comprising 20.8% of all cancers. The Cancer Genome Atlas (TCGA) divided gastric cancer to four subgroups; EBV (Epstein-Barr Virus infected, 9%), MSI (Microsatellite instability, 22%), GS (Genomically stable, 20%), CIN (Chromosomal instability, 50%). We sequenced transcriptomes of 11 gastric cancers associated with EBV infection (EBVaGC) tissues, 11 gastric cancers not associated with EBV infection (EBVnGC) tissues and 22 matched normal gastric mucosa for find unique features of Korean EBVaGC. We compared RNA expression, EBV gene expression, germline mutations, and somatic mutation between EBVaGC and EBVnGC. We found IKBKE gene, which is taking an essential role in regulating antiviral signaling pathways, expressed higher on EBVaGC tumor than EBVnGC tumor (p-value=1.73 x 10-4). While most EBV genes appeared to be similar, BMRF1,2 genes made distinctive patterns compared to previous TCGA report in expression of EBV which integrated into human cells. The BMRF-2 gene is associated with cell-to-cell spread of EBV, which is major infection mechanism of EBV. We found novel germline mutations on PABPC3, PGA3, and C4A,B in almost every samples, which are candidate for Korean specific mutations. Citation Format: Woochan Lee. EBV associated gastric cancer in Korean [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4377. doi:10.1158/1538-7445.AM2017-4377

Arsenic trioxide inhibits EBV reactivation and promotes cell death in EBV-positive lymphoma cells

BackgroundEpstein-Barr Virus (EBV) is associated with hematopoietic malignancies, such as Burkitt’s lymphoma, post-transplantation lymphoproliferative disorder, and diffuse large B-cell lymphoma. The current approach for EBV-associated lymphoma involves chemotherapy to eradicate cancer cells, however, normal cells may be injured and organ dysfunction may occur with currently employed regimens. This research is focused on employing arsenic trioxide (ATO) as EBV-specific cancer therapy takes advantage of the fact the EBV resides within the malignant cells.Methods and resultsOur research reveals that low ATO inhibits EBV gene expression and genome replication. EBV spontaneous reactivation starts as early as 6 h after re-suspending EBV-positive Mutu cells in RPMI media in the absence of ATO, however this does not occur in Mutu cells cultured with ATO. ATO’s inhibition of EBV spontaneous reactivation is dose dependent. The expression of the EBV immediate early gene Zta and early gene BMRF1 is blocked with low concentrations of ATO (0.5 nM – 2 nM) in EBV latency type I cells and EBV-infected PBMC cells. The combination of ATO and ganciclovir further diminishes EBV gene expression. ATO-mediated reduction of EBV gene expression can be rescued by co-treatment with the proteasome inhibitor MG132, indicating that ATO promotes ubiquitin conjugation and proteasomal degradation of EBV genes. Co-immunoprecipitation assays with antibodies against Zta pulls down more ubiquitin in ATO treated cell lysates. Furthermore, MG132 reverses the inhibitory effect of ATO on anti-IgM-, PMA- and TGF-β-mediated EBV reactivation. Thus, mechanistically ATO’s inhibition of EBV gene expression occurs via the ubiquitin pathway. Moreover, ATO treatment results in increased cell death in EBV-positive cells compared to EBV-negative cells, as demonstrated by both MTT and trypan blue assays. ATO-induced cell death in EBV-positive cells is dose dependent. ATO and ganciclovir in combination further enhances cell death specifically in EBV-positive cells.ConclusionATO-mediated inhibition of EBV lytic gene expression results in cell death selectively in EBV-positive lymphocytes, suggesting that ATO may potentially serve as a drug to treat EBV-related lymphomas in the clinical setting.

EBV Microrna Mir-BHRF1-2 Targets PRDM1/Blimp1: Potential Role in EBV Lymphomagenesis

PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). In DLBCL, PRDM1 is inactivated by mutations and deletions; however, there is also evidence that PRDM1 is down-regulated by microRNAs (miRNAs) in DLBCL and Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). A decrease in PRDM1 activity contributes to the pathogenesis of DLBCL and cHL by inhibiting plasma cell differentiation triggered by signal transduction pathways such as the NF-kB pathway. Since malignant EBV-positive B-cell lymphoproliferations are often associated with increased NF-kB activity, it is conceivable that abnormal PRDM1 down-regulation may play a role in their pathogenesis.EBV-positive B-cell lymphomas are postulated to originate from EBV-infected B-cells with latency III growth program of EBV gene expression. Thus, EBV-immmortalized lymphoblastoid cell lines (LCLs), which are of latency III type, serve as a good model to study EBV lymphomagenesis. We observed discordance in PRDM1 mRNA and protein levels in LCLs. By quantitative real-time reverse transcriptase PCR, PRDM1 mRNA levels in LCLs varied from 14.6% to 1259.7% relative to the multiple myeloma cell line U266, which expresses high levels of PRDM1. However, PRDM1 protein was discordantly low in LCLs compared to U266 based on immunohistochemistry and Western blotting assays, consistent with post-transcriptional regulation. EBV encodes 25 viral miRNAs, and we postulate that one of more of them may function to dampen PRDM1 expression. Indeed, a miRNA binding site containing seed match to bases 2-7 of EBV miR-BHRF1-2 was identified in positions 1565 to 1589 of PRDM1 3’ untranslated region. MiR-BHRF1-2 functionally targeted this specific binding site and repressed luciferase reporter activity. Mutation in the seed region of this site relieved the repression in comparison to the wild type control.MiR-BHRF1-2 was highly expressed in LCLs, while it was barely detectable in the EBV-positive Burkitt lymphoma cell line MUTU I, which has latency type I. Importantly, immunoblotting assay demonstrated an up-regulation of PRDM1 protein level in CCL156 and CCL159 LCL cells transfected with miR-BHRF1-2 inhibitor relative to those transfected with miRNA Inhibitor negative control, supporting a role of miR-BHRF1-2 in PRDM1 down-regulation in vivo. To examine the biological consequences of increased PRDM1 expression in LCL cells, PRDM1 was over-expressed in JY25 and CCL159 LCL cell lines. Enforced expression of PRDM1 induced apoptosis in both cell lines. Furthermore, bromodeoxyuridine (Brdu) incorporation study demonstrated that overexpression of PRDM1 reduced the percentage of S phase from 43.4% to 27.6% in CCL159 cells, and 39.5% to 27.9% in JY25 cells, respectively. Whole transcriptome sequencing (RNA-seq) identified a set of potential PRDM1 direct target genes whose expressions decreased in both LCL cell lines upon PRDM1 over-expression. These genes have broad functions including cell proliferation and survival, transcription and translation, mitochondrial functions, and cytoskeleton. Although no significant changes in cell cycle and apoptosis were observed upon transfection of miR-BHRF1-2 inhibitor, RNA-seq analysis of CLL159 cells transfected with miR-BHRF1-2 inhibitor revealed a small subset of repressed genes which overlapped with those identified by PRDM1 over-expression. This finding suggests that the increase in PRDM1 expression upon miR-BHRF1-2 inhibition, albeit small, is capable of repressing a subset of PRDM1 target genes with potential biological effects.In summary, our findings demonstrate that PRDM1 is a target of EBV miR-BHRF1-2. MiR-BHRF1-2 mediated PRDM1 down-regulation may contribute to the pathogenesis of EBV-associated B-cell lymphomas by inhibiting the transcription repression program of PRDM1 and limiting PRDM1-mediated cellular changes detrimental to tumor growth, including cell cycle arrest and apoptosis. Further characterization of the target genes whose expression is up-regulated by miR-BHRF1-2-mediated PRDM1 down-regulation may provide important clues to the pathogenetic function of miR-BHRF1-2 and EBV oncogenesis in general. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3425: Whole-transcriptome analyses of EBV-associated nasopharyngeal carcinoma using next-generation transcriptome sequencing

Abstract Nasopharyngeal carcinoma (NPC) is unique among epithelial cancers arising from the head and neck regions. This specific cancer type is consistently associated with EBV infection. Using RNA-seq, we have conducted a comprehensive assessment of EBV and cellular gene expression in six NPC cell line and patient-derived xenografts (PDXs). In these tumor lines, expression of EBV latent genes including EBNA1, LMP1, LMP2A and BamH1 A transcripts was quantitated. Among the viral genes, the BamH1 A transcripts were detected as the major transcripts with various splicing variants. Expression of these transcripts in NPC was further validated by qRT-PCR analysis. Active lytic gene expression and lytic viral replication was not observed in these tumor lines. Analysis of the RNA-seq data has identified multiple fusion transcripts. A total of 18 candidate fusions were validated in these tumor lines. A novel fusion transcript, UBR5-ZNF423 was detected in C666-1 cells and 8.3% of primary tumors. The transcript produces a chimeric fusion protein which might alter the function of EBF transcription factor. A significant growth inhibitory effect was observed in UBR5-ZNF423 knockdown C666-1 cells. Constitutive expression of UBR5-ZNF423 fusion in NIH3T3 cells induced in vivo tumor formation in nude mice model. The findings strongly suggest that the fusion gene play a causative role in NPC tumorigenesis. Using the expression profile of immortalized nasopharyngeal epithelial cells as control, our study also revealed the differential expression of cellular genes and specific alternative spliced transcripts in NPC. Significant upregulation of genes involved in cytokine-cytokine receptor interaction (e.g. CCL5, CCL20, CXCL10, CXCR4, IL2RG) was consistently detected in the tumor lines. Most of them were targets of the NF-kappaB signaling pathways, which are constitutively activated in EBV-associated NPC. In addition, the analyses also discovered several potential druggable targets (e.g. CD74) and oncogenic non-coding RNAs (e.g. AFAP1-AS1) in NPC, which may contribute to the development of new therapeutic strategies for this cancer. The whole-transcriptome analyses greatly enhance our understanding of the molecular basis of EBV-infected NPC. Citation Format: Cathy Ka-Yan Mak, Grace Tin-Yan Chung, Kevin Yuk-Lap Yip, Ken Kai-Yuen Tso, Sau-Dan Lee, Siu-Tim Cheung, Sai-Wah Tsao, Pierre Busson, Ka-Fai To, Kwok-Wai Lo. Whole-transcriptome analyses of EBV-associated nasopharyngeal carcinoma using next-generation transcriptome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3425. doi:10.1158/1538-7445.AM2014-3425

Nucleolin is important for Epstein–Barr virus nuclear antigen 1-mediated episome binding, maintenance, and transcription

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1's N-terminal 100 aa and NCL's RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K429A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention.

A phase IB trial of 5-azacitidine (5AC) and suberoylanilide hydroxamic acid (SAHA) in patients with metastatic or locally recurrent nasopharyngeal carcinoma (NPC) and NK-T cell lymphoma.

e17017 Background: Epigenetic up-regulation of EBV and cellular genes via demethylation and histone deacetylase inhibition can induce EBV lytic replication enhancing immune mediated tumor killing and up-regulation of tumor suppressor genes resulting in tumor apoptosis. Methods: Patients (Pt) with relapsed or refractory NPC and NK-T cell lymphomas were enrolled to determine safety, tolerability, pharmacokinetics (PK), pharmacodynamics and preliminary anti-tumor activity using a dose escalation design. 5AC was administered on days 1 to 10 sub-cutaneously while SAHA was administered on days 1 to 14 orally. PK for SAHA, EBV viral load, characterization of circulating EBV, Immunohistochemistry (IHC) and EBV promoter methylation analysis in tumor tissue were performed. Results: 11 pt have been treated (M:F 8:3, median age 48, R: 35-71) at 3 dose levels – 5AC 50 mg/m2 and SAHA 200 mg b.i.d. (dose level 1), 5AC 37.5 mg/m2 and SAHA 200 mg q. am and 100 mg q. pm (dose level 2), and 5AC 25 mg/m2 and SAHA 100 mg b.i.d (dose level 3). Median number of previous treatment regimens was 3 (R:1-6). Dose limiting toxicities (DLT) were seen in 2/2 pts at dose level 1: grade 4 thrombocytopenia (1 pt), grade 3 nausea, vomiting and fatigue (2 pts), and grade 5 hepatic failure (1 pt). Two of six patients at dose level 2 experienced DLT: grade 3 fatigue (1 pt) and worsening of pre-existing Sweet’s Syndrome (1 pt). Common AEs (G1/2) included fatigue (73%), cough (64%), anorexia (55%), and injection site reaction (45%). One minor response was seen and 5 pt had prolonged stable disease (>16 weeks), including one patient for 88 weeks. Analysis of post-treatment tumor biopsies showed demethylation of EBV lytic cycle gene promoters after treatment. SAHA PK, IHC results for EBV gene expression in tumor tissue, EBV viral load and characterization of circulating EBV will be presented. Conclusions: 5AC/SAHA appears to be tolerable at dose level 3 with suggestion of clinical benefit. Analysis of post-treatment tumor and blood samples suggests that modulation of EBV gene expression may play a role in the mechanism underlying clinical benefit. Continued accrual at dose level 3 is ongoing. Clinical trial information: NCT00336063.

Different Patterns of Epstein-Barr Virus Latency in Endemic Burkitt Lymphoma (BL) Lead to Distinct Variants within the BL-Associated Gene Expression Signature

Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as "molecular BL" (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.

CD44+ cells have cancer stem cell–like properties in nasopharyngeal carcinoma

A subpopulation of cells within a tumor appears to have the exclusive ability to initiate tumors, self-renew, and differentiate. These "cancer stem cells" (CSCs) are CD44(+) in several epithelial malignancies. We examined the potential of CD44 to identify the CSC population in nasopharyngeal carcinoma (NPC). C666, an Epstein-Barr virus-positive (EBV(+) ) human NPC cell line, was stained for CD44 and sorted by fluorescence-activated cell sorting (FACS). CD44(+) and CD44(-) subpopulations were evaluated for (1) proliferative potential, (2) ability to differentiate, (3) expression of markers of epithelial-to-mesenchymal transition (EMT) and EBV genes, and (4) the ability to initiate tumors in vivo. Immunocompromised mice were injected with CD44(+) and CD44(-) populations to assess the tumor-initiating capacity. Immunohistochemistry for CD44 was performed on an 87-patient tissue microarray (TMA), and clinical correlations were examined. Heterogeneous expression of CD44 was seen among C666 cells. CD44(+) cells differentiated into CD44(-) cells, indicating a hierarchical relationship. Further, CD44(+) cells exhibited a more robust tumor-initiating capacity in the xenograft model. However, no differences were seen in proliferation rates in vitro, EBV gene expression, or expression of EMT markers between CD44(+) and CD44(-) subsets. Patient tumors were heterogeneous for CD44 staining, and a trend toward an association between CD44 expression and clinical outcome was observed. NPC contains a CD44(+) subpopulation with features consistent with CSCs. There was a trend toward an association between CD44 expression within NPC tumors and decreased time to local failure/relapse in patients.

Expression of Epstein-Barr Virus Gene and Clonality of Infiltrated T Lymphocytes in Epstein-Barr Virus-associated Gastric Carcinoma

BackgroundEpstein-Barr virus associated gastric lymphoepithelioma-like carcinoma (LELC) is characterized by the intensive infiltration of lymphoid cells, the presence of EBV, and the better prognosis over typical adenocarcinoma. Thus, it was assumable that viral latent proteins may be responsible for the recruitment of a certain T cell repertoire to EBV-associated gastric carcinoma.MethodsTo examine above possibility, EBV gene expression in gastric carcinoma tissues and usage of TCR among the tumor infiltrating lymphocytes were analyzed.ResultsEBV specific DNA and EBERs RNA were detected in 4 out of 30 patients. RT-PCR analysis revealed that all 4 of EBV-positive tumor tissues expressed EBNA1 mRNA and BARTs and LMP2a was detected only one sample out of 4. However, the EBNA2 and LMP-1 transcripts were not detected in these tissues. CD8+ T cells were the predominant population of infiltrating lymphocytes in the EBV-positive gastric carcinoma. According to spectra type analysis of infiltrating T cells, 10 predominant bands were detected by TCR Vβ CDR3 specific RT-PCR from 4 EBV-positive tumor tissues. Sequence analysis of these bands revealed oligoclonal expansion of T cells.ConclusionThese findings suggest that clonally expanded T cells in vivo might be a population of cytotoxic T cells reactive to EBV-associated gastric carcinoma.

Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle Induction with Bortezomib Is a Response to ER Stress

AbstractAbstract 1736EBV and KSHV are associated with a variety of lymphoid malignancies. Bortezomib, a proteasome inhibitor, is among the most potent activators of EBV and KSHV lytic infection and is active at nanomolar concentrations. Studying EBV Burkitt's cell lines, KSHV primary effusion lymphoma cell lines and EBV myeloma cell lines, we found that lytic gene expression is a class effect shared by other proteasome inhibitors, such as MG-132 and epoxomicin. Proteasome inhibition results in IKB stabilization and inhibition of NFKB activity and this effect has been implicated in many of the effects of bortezomib. We studied the promoters of EBV and KSHV immediate early viral protein reporters (EBV Zta and KSHV Rta) and found that IKB super-repressor did not activate promoter reporters suggesting that other pathways must be important in activation. Therefore, we sought to better understand the drug's impact on viral gene expression in virus-associated tumor cells. ER stress has been implicated in bortezomib's antitumor effects. Thapsigargin and tunicamycin are classic activators of ER stress that do not act through proteasomal inhibition. These agents were found to be potent activators of the EBV and KSHV lytic cycle. Previous studies have identified C/EBP family members (C/EBP alpha and beta) as activators of EBV and KSHV immediate early gene expression. In other studies, C/EBP family members (C/EBP beta, CHOP10) have been implicated in regulating the ER stress response. We found that bortezomib, thapsigargin and tunicamycin increased levels of the activating C/EBP beta LAP isoform as assessed by immunoblot and by real-time RT-PCR. Treatment also led to increase in ATF4, XBP1(s), CHOP10, and ATF6 cleavage, all consistent with induction of the ER stress response. Additionally, we showed that treatment with bortezomib increased C/EBP beta binding to previously characterized binding sites in the Zta promoter and expression of C/EBP beta LAP isoform was sufficient to activate EBV immediate early lytic promoters. Finally, we demonstrated that in tumor cell lines with C/EBP beta silenced by doxycycline regulated siRNA, induction of EBV lytic induction by bortezomib and thapsigargin was blocked. These results demonstrate that both human lymphoma associated herpesviruses (EBV and KSHV) are activated into lytic cycle by bortezomib and that these effects are mediated through ER stress pathways and specifically involve C/EBP beta. Disclosures:No relevant conflicts of interest to declare.

C/EBPbeta mediates bortezomib-induced EBV and KSHV lytic gene expression

Bortezomib is a proteasome inhibitor used clinically for the treatment of multiple myeloma and mantle cell lymphoma and is being evaluated for the treatment of AIDS-related lymphoma and Kaposi’s sarcoma in the AIDS Malignancy Consortium. Activation of herpes zoster infection is a well-recognized complication of bortezomib therapy. In previous work, we have shown that bortezomib is a potent inducer of EBV and KSHV lytic gene expression and demonstrated a novel approach to therapy that leverages this activation to facilitate tumor imaging and treatment. In an effort to better understand the relevant pathways, we confirmed that bortezomib at 20 nM concentrations induces expression of EBV and KSHV immediate early genes (EBV ZTA, KSHV RTA) as assessed by immunoblot and by reverse transciptase PCR for the associated transcripts. Bortezomib activates EBV ZTA promoter expression and KSHV RTA promoter expression in reporter assays. C/EBPalpha and beta protein family members are recognized activators of EBV ZTA and KSHV RTA expression. Bortezomib does not alter C/EBPalpha protein level as assessed by immunoblot but does lead to increased C/EBPbeta. C/EBPbeta includes activating isoforms (LAP) and an inhibitory isoform (LIP). In reporter assays, we show that C/EBPbeta LAP activates the EBV promoters whereas LIP is inhibitory. In ChIP assays we show that bortezomib treatment leads to increased C/EBPbeta binding to the ZTA promoter. Mutation of the binding sites abolishes activation of the promoter associated with bortezomib treatment. In order to determine whether C/EBPbeta expression simply tracks with lytic viral gene expression or plays a key role in mediating such expression, we created a tet-regulated C/EBPbeta knockdown. In the presence of doxycycline, C/EBPbeta is suppressed, and bortezomib lytic induction of EBV as measured by increases in ZTA mRNA, ZTA protein, and viral DNA copy number are all blunted. Induction of lytic viral gene expression in EBV Akata cell lines by anti-Ig treatment is also blunted. These results suggest that C/EBPbeta family members play a key role in mediating activation of EBV and KSHV lytic infection following bortezomib and other inducers.1

Epstein-Barr Virus-Associated Haemophagocytic Lymphohistiocytosis in Adults Characterised by High Viral Genome Load within Circulating Natural Killer Cells.

Abstract 3672Poster Board III-608 IntroductionEpstein Barr virus (EBV) is predominantly B lymphotrophic both in-vitro and in vivo, where in immunocompetent individuals the virus persists asymptomatically in the B lymphoid compartment under host T cell control. EBV's association with B cell malignancies, such as Hodgkin and Burkitt lymphoma, can be viewed as rare accidents of the virus' lifelong interaction with the B cell system. By contrast, EBV infection of NK and T cells is considered a rare event but is nonetheless strongly associated with a spectrum of rare lymphoproliferations: EBV-associated haemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV), aggressive NK leukaemia and NK/T lymphoma all characterised pathogenically by the presence of monoclonal EBV in the T and/or NK cells. The mechanism of viral entry and its contribution to lymphoproliferation in these cell lineages remains to be clearly defined. The majority of reported cases of EBV-HLH occur in the context of primary EBV infection in children or adolescents, some of whom have a defined inherited immune defect. Adult cases of EBV-HLH occur extremely rarely and appear to be more frequent in individuals of East Asian origin. Furthermore, the vast majority of analysed cases of EBV-HLH have identified CD8+ lymphocytes as the predominant virus-bearing cell. To-date, EBV infection of (CD3-CD56+) circulating NK cells has not been reported and the pattern of viral gene expression remains unclear. Methods/PatientsWe analysed peripheral blood from three consecutive cases of EBV-HLH, referred to our laboratory between 2007-2009, to identify the predominant virus-harbouring cell. All three cases occurred in adults (mean age 44yrs), with no history of inherited immunodeficiency, who presented with clinical and laboratory features consistent with a diagnosis of HLH; fever, hepatosplenomegaly, pancytopenia, markedly elevated serum ferritin and lactate dehydrogenase and EBV copy number of 105-106 per millilitre of whole blood. Haemophagocytosis was unequivocally present on tissue biopsy from two patients. Mononuclear cells were separated using the MoFlo™ cell sorter into pure populations. Patient 1 and 2: CD19+CD3 −CD56−, CD3+CD19−CD56−, and CD56+CD3−CD19−. Patient 3: CD19+CD3 –CD16−, CD3+CD19−CD16−, CD16+CD3−CD19− and CD3−CD19−CD16−. DNA was subsequently extracted from each population and assayed by quantitative PCR, expressed as genome copies per million cells. ResultsIn all three cases we found the predominant EBV load within the non-B, non-T lymphocyte populations; definitively shown to be the CD56+CD3− cell fraction in 2 cases and for case 3 within CD3−CD19−CD16− lymphocytes likely to represent CD56+CD16- NK cells (a minority population in normal peripheral blood). A representative figure is shown: [Display omitted] We then quantitatively examined latent and lytic viral gene transcripts by real-time PCR and, in contrast to previously published data, we found a tightly restricted pattern of EBV gene expression with extremely high levels of EBER (EBV-encoded RNA) transcripts present. Lymphocytes derived from tonsillar tissue and peripheral blood, from both healthy and immunosuppressed individuals, served as control samples and demonstrated the predominant EBV genome load in the CD19+ B-cells but not the T or NK fractions. ConclusionThis novel finding of high EBV genome copy numbers and a restricted pattern of viral gene expression, within circulating natural killer cells in the context of adult EBV-HLH, is both pathogenically intriguing and importantly, has relevance for the investigation of targeted therapies for this aggressive disease. Disclosures:No relevant conflicts of interest to declare.