EBV-Associated Lymphoma Consortium Annual Meeting (Abstract)

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10 150870 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #10 db31f8c0-6f4e-42b6-86fd-eda10b07b1f8 No Wed, 09/03/2025 - 21:13 Wed, 09/03/2025 - 21:13 Wed, 09/03/2025 - 21:13 Anonymous 10.208.24.230 Isabella Kong PhD Postdoctoral Fellow Weill Cornell Medicine New York New York iyk4001@med.cornell.edu Spatial molecular imaging of EBV+ HIV-associated lymphomas reveals divergent cytokine expression and tumor:immune niches linked to EBV latency program Epstein-Barr virus (EBV) persists in tumors in distinct latency states: latency I (expressing only EBNA1) and latency II-III (expressing immunogenic proteins such as LMP1/2 and/or EBNA2/3, targeted by cytotoxic T cells (CTLs)). We previously reported that latency restriction is regulated by DNA methyltransferase 1 (DNMT1) (Guo et al, Nat Microbiol 2020), and that treatment with decitabine induces latency II/III antigen expression in latency I tumors, sensitizing them to EBV-specific CTLs (Dalton et al, Blood 2020). Beyond latency, DNMT1 also controls cytokine expression by repressing the expression of cytokines (IL1b, IL6, IL4, TNFa) and chemokines (CXCL9, CXCL10). We hypothesized that latency programs generate distinct cytokine milieus, shaping tumor-immune interactions and facilitating immune evasion.
To test this, we compared cytokine expression in latency I and III cell lines and performed spatial molecular imaging in EBV+ HIV-associated lymphomas. DNMT1 was reduced in latency III cells, which produced more IL4, IL6, IL10 and IL21 than latency I cells. To assess heterogeneity in vivo, we performed imaging mass cytometry on 53 EBV+ HIV-associated lymphomas (Burkitt lymphoma, primary effusion lymphoma, diffuse large B cell lymphoma, Hodgkin lymphoma, polymorphic lymphoproliferative disorder and plasmablastic lymphoma). Tumors displayed striking intratumoral heterogeneity, with most cells in latency I but variable subsets in latency IIa, IIb, and III. Consistent with cell line data, latency III tumor cells exhibited elevated IL4, IL6, IL10 and Il21 expression. Neighbourhood analysis revealed that latency I cells were preferentially surrounded by macrophages, CD4+ and CD8+ T cells, whereas latency III cells were enriched for NK cells, dendritic cells and exhausted LAG3+ CD8+ T cells.
In summary, latency III cells show reduced DNMT1, increased cytokine expression, and distinct immune niches, suggesting that EBV latency programs shape the tumor-immune niche and contribute to immune evasion.
2 149987 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #2 26f0bb87-964e-4198-b235-fb2b0bb26c4c No Wed, 08/27/2025 - 11:58 Wed, 08/27/2025 - 11:58 Wed, 08/27/2025 - 11:58 Anonymous 10.208.24.239 Kelsey Smith B.S. Research Specialist University of Wisconsin-Madison Madison Madison kasmith26@wisc.edu The role of LMP2A on pathogenesis of EBV-driven lymphomas in vivo One major focus of this project is to investigate the impact of LMP2A on the pathogenesis of EBV-driven lymphoma using a "humanized" mouse xenograft model. We compared mice transplanted with cord blood mononuclear cells infected with WT or LMP2A-deleted (ΔLMP2A) EBV (M81 strain). Similar to published findings, we observed that ΔLMP2A and WT initially led to similar tumor size and incidence. However, ΔLMP2A tumors failed to progress in size over time and were much less invasive, significantly decreasing mortality in those mice. In both WT and ΔLMP2A mice we observed two distinct subsets of B cells: plasmablastic (CD38hi/CD20lo) and less differentiated CD20hi/CD38lo. In pre-invasive tumors, plasmablastic cells formed capsule-like structures at the leading edge of the tumor, while the less differentiated cells clustered at the center. As tumors progressed, plasmablastic cells were the first to invade organ tissue. These findings led us to further investigate the invasive quality of these plasmablastic cells.
In RNA-Seq analysis of LCLs controlled for BCR isotype to isolate the effect of LMP2A, we found that WT LCLs upregulated many genes associated with plasma cell differentiation compared to ΔLMP2A LCLs. Published work has established a gradient of proliferation and differentiation in LCLs and identified LMP1 as the driver of proliferation. We hypothesize that LMP2A is responsible for differentiation. We believe this gradient can be explained by the bi-directional ED-L1 promoter, which drives expression of both LMP1 and LMP2B. When ED-L1 activity is high, LMP1 drives proliferation and the NF-kB pathway, and LMP2B diminishes signaling activity of LMP2A, leading to decreased differentiation. While these results clarify the impact of LMP2A on plasmablastic cell differentiation, the factor(s) regulating invasion remain unknown. Ongoing studies aim to identify the switch that regulates plasmablastic cell invasion.
3 150046 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #3 0573b5ec-04a8-458e-bf34-00f0b8ab1387 No Wed, 08/27/2025 - 16:49 Wed, 08/27/2025 - 16:49 Wed, 08/27/2025 - 16:49 Anonymous 10.208.28.150 Shannon C. Kenney MD WARF Professor of Oncology and Medicine University Of Wisconsin School Of Medicine And Public Health Madison Madison skenney@wisc.edu Epstein-Barr virus, germinal center B cell, LMP2A, DLBCL, Plasmablastic lymphoma Epstein-Barr virus (EBV) infection causes human Germinal Center B cell-derived lymphomas in the absence of EBNA2 expression EBV causes human B-cell lymphomas, including Burkitt lymphomas (BLs), diffuse large B cell lymphomas (DLBCLs), Hodgkin lymphomas (HLs) and Plasmablastic lymphomas (PLs). EBV+ lymphomas in immunocompetent humans are usually derived from germinal center (GC)-experienced B cells and have stringent latency forms that do not express the viral transforming protein, EBNA2. EBV+ lymphomas with “Type II” latency (the most common latency type in DLBCLs and HLs) are driven by the LMP1 and LMP2A EBV proteins, which activate NF-KB and B-cell receptor-like signaling, respectively. However, there is currently no model system for studying how type II latency transforms normal human GC-derived B cells into lymphomas in vivo. Here we show that tonsil GC B cells (GCBs) infected with an EBNA2-deleted EBV mutant (ΔEBNA2) proliferate indefinitely on a CD40L/IL21-expressing feeder layer and form lymphomas in NSG mice that resemble human DLBCLs and PLs. ΔEBNA2-infected tumors that have high-level LMP2A expression (with type II latency) form lymphomas that express plasmablast markers, while tumors with LMP2A-negative “Wp-restricted” latency (a latency type found in some EBV+ human BLs) have a GCB DLBCL-like phenotype. Using this novel model system, we show that LMP2A activates expression of cellular proteins required for plasmablast differentiation and decreases expression of cellular proteins required for GCB and B-cell identity. Furthermore, LMP2A increases expression of genes involved in lymphocyte mobility and trafficking and enhances tumor invasiveness in vivo. This new model system can thus be used to define roles of viral and cellular proteins in EBV-induced human GCB-derived lymphomas that lack EBNA2 expression
8 150851 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #8 d63c82e4-4280-4105-a704-72d82c0756df No Wed, 09/03/2025 - 17:41 Wed, 09/03/2025 - 17:41 Wed, 09/03/2025 - 17:41 Anonymous 10.208.28.30 Sumita Bhaduri-McIntosh M.D., Ph.D. Professor University of Florida Gainesville Gainesville sbhadurimcintosh@ufl.edu DLBCL, Synthetic lethality, polymerase theta, DNA repair, PROTAC Engineering PROTACs to degrade polymerase theta With EBV+ DLBCL associated with poorer outcomes compared to EBV- DLBCL, we are focusing on EBV-associated mechanisms, in particular, repair of cellular DNA, an underexplored area. We have previously shown that EBV+ cancer cells lack the ability to repair toxic DNA double-strand breaks (DSB) by homologous recombination, the most error-free form of repair – thereby becoming susceptible to synthetic lethal (SL) therapies that target alternative mechanisms of DSB repair such as microhomology-mediated end-joining (MMEJ).

Our experiments demonstrate that EBV-transformed cells, EBV-cancer lines, EBV+ DLBCL xenografts, and primary EBV+ DLBCLs from AIDS patients express higher levels of polymerase theta compared to peripheral mononuclear cells and AIDS-related EBV- DLBCLs; typically, polymerase theta (POLθ) is the enzyme responsible for MMEJ-mediated repair of DSBs. We find that EBNA1 drives higher expression of POLθ in EBV+ tumors and that POLθ indeed facilitates DSB repair and contributes to cellular DNA replication and survival of EBV+ tumor cells. POLθ inhibitors (targeting the helicase or polymerase domain), alone or in combination with other agents, are in clinical trials, though in pre-clinical models, such inhibitors display biologic effects at tens of micromolar concentrations. Using PROTAC technology, we have developed small molecule degraders that degrade POLθ at nanomolar concentrations. In EBV-cancer lines, our lead compound, 23, degrades ≥90% of the intracellular POLθ at ≤10nM concentration, while the negative control compound (warhead), unable to bind E3 ligase, does not. As expected, compound 23 degrades POLθ via the ubiquitin-proteasome system. Across EBV-cancer cell lines, compound 23 consistently kills cells at concentrations that are up to 1000-fold lower than the prototypic POLθ inhibitor Novobiocin that is undergoing clinical trial testing. Following rigorous testing and lead optimization, we expect to translate the use of a POLθ PROTAC, alone or with other drugs, to cancers that demonstrate defects in DNA repair and/or reliance on POLθ.
9 150860 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #9 9271ea5a-e361-47d8-9672-ef883fae9f73 No Wed, 09/03/2025 - 18:20 Wed, 09/03/2025 - 18:20 Wed, 09/03/2025 - 18:20 Anonymous 10.208.28.30 Michael T McIntosh Ph.D. Professor Pediatrics and Molecular Genetics and Microbiology University of Florida GAINESVILLE GAINESVILLE mmcintosh@peds.ufl.edu Synthetic lethal, Epstein-Barr virus, EBV, diffuse large B cell lymphoma, DLBCL Single nucleus RNA-seq of EBV-positive AIDS-DLCBLs Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma and the leading cause of cancer-related death in HIV-infected individuals. Up to 40% of individuals have refractory or relapsing DLBCL, and associated Epstein-Bar virus (EBV) coincides with even higher rates of mortality. Earlier, we showed that EBV activates Signal transducer and activator of transcription 3 (STAT3) leading to loss of the DNA damage response checkpoint and homologous recombination repair. While this ensures proliferation despite damage, it also increases reliance on alternate dsDNA break repair mechanisms. Targeting such parallel pathways represents a promising new approach to treatment known as synthetic lethality. Here, we investigate the gene expression profiles of EBV+-DLBCL tumors from HIV positive individuals to identify additional codependent DNA replication/repair pathways that may be targetable by synthetic lethal strategies. Five EBV+-DLBCL biopsies along with two matched normal tissue controls were obtained from the AIDS Cancer Specimen Resource. Immunohistochemistry confirmed the presence of CD20+ cells, EBV’s LMP-1, and proliferating cells marked by Ki67. Single nuclei suspensions were isolated from the frozen samples and subjected to barcoded cDNA amplification and sequencing using the PARSE single-nucleus RNA-seq protocol. Sequencing was performed on an Illumina NovaSeq to a depth of ~50,000–200,000 reads per nucleus. Only nuclei with >100 detected genes and < 15% mitochondrial gene content were retained for analysis. Data were processed by log-normalization and analyzed by shared near-neighbor and UMAP in Seurat. Analysis revealed varying cell types within and between the different samples as well as different degrees of malignant B cells. While ongoing, highly expressed genes enriched in malignant B cell populations varied greatly across samples. In addition to anticipated oncogenes, comparisons across samples and between the tumor tissues and matched normal control tissues reveal several genes involved in DNA replication, repair or metabolism.
1 149570 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #1 4c14a1c5-4121-44d7-8592-4f5bb9ae7215 No Sat, 08/23/2025 - 05:15 Sat, 08/23/2025 - 05:15 Sat, 08/23/2025 - 05:15 Anonymous 10.208.28.6 Ting-Ting Wu PhD Professor UCLA Los Angeles Los Angeles twu@mednet.ucla.edu Strategically Engineered Multi-Antigen KSHV Vaccine: Integrating Humoral and Cellular Immunity for Effective Protection Infection of Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8), is estimated to account for 34,000 new cancer cases each year, presenting a significant healthcare challenge globally. Typically, KSHV infection in healthy infected individuals results in weak neutralizing antibody responses and low T cell immunity. This modest immunity during natural infection calls for a vaccine that can circumvent the immune evasion mechanisms of KSHV, effectively priming the immune system to generate robust and reliable immunity across individuals. Such a vaccine would not only protect against initial infections but also help individuals maintain better control over persistent infection, thereby reducing the risk of developing KSHVassociated diseases. This is particularly vital for high-risk populations, including individuals with an increased likelihood of HIV-1 infection, transplant patients receiving immunosuppressive therapy, and people living in resource-limited endemic regions of Africa. Our proposal addresses this critical medical need.
Our proposal outlines a comprehensive and innovative vaccine strategy against KSHV, harnessing antibody and T cells to establish strong antiviral immunity. Aim 1 focuses on generating potent humoral antibody responses through structure-guided immunogen design, generating neutralizing and non-neutralizing activities that block infection and mediate the immune clearance of infected cells. Aim 2 is dedicated to enhancing vaccineinduced T cell immunity targeting latently infected cells by utilizing innovative RNA-based adjuvants. These RNA-adjuvanted T cell responses are designed to complement antibody-mediated antiviral effects. Aim 3 seeks to integrate these strategies by combining vaccine components that simulate both antibody and T cell responses while maintaining the immunogenicity of each antigen. This aim will also employ mice reconstituted with the human immune system to address species differences in terms of dendritic cell activation and inflammatory responses after receiving our combined vaccine.
6 150781 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #6 038de749-a6fb-45be-9e21-4f14b388861b No Wed, 09/03/2025 - 13:40 Wed, 09/03/2025 - 13:40 Wed, 09/03/2025 - 13:40 Anonymous 10.208.28.30 Josselyn Pena phD Postdoc Stanforf University Stanford Stanford smkrams@stanford.edu EBV, NK cells, NKG2A+ NK Cell Cytotoxicity of Epstein-Barr Virus Infected B Cells is Mediated Through the NKG2D and NKp30 Activating Receptors Epstein-Barr virus (EBV) is a human γ-herpesvirus that establishes latency and lifelong infection in B cells. Failure to control latent EBV infection can result in a variety of malignancies, including post-transplant lymphoproliferative disease (PTLD) in transplant recipients. Studies have implicated natural killer (NK) cells as critical in the host defense against EBV infection.

To understand the relationship of human NK cells and EBV in vivo, we utilized mass cytometry to analyze circulating NK cells in children that are seropositive (n=11) or seronegative (n=6) for EBV. After gating on CD3-NKp46+ NK cells, clustering resulted in 10 distinct subpopulations. Higher proportions of NKG2A+ NK cell subsets were detected in the circulation of EBV-seropositive children as compared to seronegative children.

NKG2A recognizes non-classical HLA-E on target cells; we determined that HLA-E expression was markedly increased in EBV-infected cells compared to autologous uninfected B cells. To understand the NKG2A:HLA-E interactions in EBV-infected B cells, we generated HLA-E knockout (KO) EBV+ lymphoblastoid cell lines (LCL) using CRISPR/Cas9 gene editing and observed a significant increase (p <0.001) in killing of the KO EBV+ LCL by autologous and allogeneic NK cells as compared to wild-type EBV+ LCL or mock transfected EBV+ LCL. Moreover, increased killing of EBV+ LCL was also observed in the presence of anti-NKG2A as compared to untreated or isotype controls (p < 0.01). EBV-responsive NK cells express high levels of the NKp30 and NKG2D activating receptors, and ligands that bind to NKG2D (MICA/MICB and ULBP) and NKp30 (B7-H6) are increased on EBV+LCL after coculture with NK cells. Disruption of the NKG2A:HLA-E immune checkpoint axis, either through the decrease of HLA-E on target cells or the more clinically relevant blocking of NKG2A on NK cells, enhances killing of EBV-infected B cells.
5 150772 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #5 a3c7d132-125a-4b6c-95a6-80d03a9430f9 No Wed, 09/03/2025 - 13:28 Wed, 09/03/2025 - 13:28 Wed, 09/03/2025 - 13:28 Anonymous 10.208.24.230 Ashley Haluck-Kangas PhD Post-Doctoral Fellow Stanford University School of Medicine Stanford, CA Stanford, CA ahaluckk@stanford.edu EBV, LMP1, PTLD Characterization of Latent Membrane Protein 1 Diversity in EBV+ Post Transplant Lymphoproliferative Disease EBV is associated with the development of Post-Transplant Lymphoproliferative Disease (PTLD) in immunosuppressed solid organ and hematopoietic stem cell recipients. Prior studies from our group identified mutations (G212S and S366T) in latent member protein 1 (LMP1) of EBV that were associated with EBV+ PTLD (OR=11.7). However, some matched controls also carried both LMP1 mutations and did not develop EBV+ PTLD. Therefore, we analyzed LMP1 in EBV+ PTLD cases with the G212S and S366T mutations (n=28), EBV-seropositive transplant controls without PTLD that also have the two LMP1 mutations (n=48), and EBV-seropositive transplant controls without the LMP1 mutations (n=11) to determine if other non-synonymous changes were associated with EBV+ PTLD. We identified three highly mutated sites in EBV+ PTLD LMP1 (S309N, Q334R, and L338S or L338P), however these amino acid changes were also seen at similar frequencies in LMP1 from controls. Patients with EBV+ PTLD had fewer non-synonymous changes (mean 7.57, SE 0.551) in LMP1 compared to controls (mean 9.08, SE 0.506), and there were no novel non-synonymous changes in LMP1 associated with EBV+ PTLD. We also compared the frequency of the well-described 10 amino acid deletion in LMP1 and while the deletion was more common in LMP1 from EBV+ PTLD cases, there was no significant difference in the deletion frequency in cases and controls. Finally, we analyzed the HLA-E*01:03 epitope (GGDPHLPTL) previously characterized by our group to strongly bind and stabilize HLA-E. We did not observe any significant enrichment of nonsynonymous mutations in the LMP1 sequence encoding this motif in cases or controls. Our data demonstrates that considerable genetic variability exists in the c-terminal domain of LMP1. However, no specific variation appears to predict the development of EBV+ PTLD. It is possible that predictive and/or functional genetic variants may occur in other EBV genes which is the focus of on-going studies.
7 150825 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #7 263ffdff-7bc7-4e6c-bdb1-850fcc17ecd0 No Wed, 09/03/2025 - 15:15 Wed, 09/03/2025 - 15:15 Wed, 09/03/2025 - 15:15 Anonymous 10.208.24.230 Edward Vizcarra PhD Post-doctoral Fellow Stanford University School of Medicine Stanford, CA Stanford, CA eav91@stanford.edu EBV, T cells Functional alterations in CD4+ and CD8+ T cells are associated with persistent EBV DNAemia in transplant recipients EBV is associated with the development of post-transplant lymphoproliferative disease (PTLD), a serious complication of solid organ and bone marrow transplantation. To understand the immune response to EBV in transplant recipients we analyzed the T cell response in EBV seropositive transplant recipients with no evidence of EBV disease and in transplant recipients with chronic (3-6 months) EBV DNAemia (CED). We detected a significantly reduced proportion of IL-2/IFN-g polyfunctional CD4+ T effector memory (TEM) cells in the CED group compared to the controls. CED CD8+ T cells had a higher proportion of TEMRA cells but significantly fewer of these were IL-2+TEMRA cells. Similarly, there were fewer IL-2+, TNF-a+, and IL-2/TNF-a+ polyfunctional TEM CD8+ T cells in the CED group compared to controls. Moreover, the CED group had fewer clonally expanded TEMRA compared to the control group. To investigate the molecular underpinnings of these differences we performed scRNAseq on PBMC after stimulation with EBV latent cycle peptides. Using UMAP we identified 19 clusters that include naïve, transitional, TEM, TEMRA, and central memory CD8+ T cells. CED CD8+ TEMRA and TEM cells express higher levels of exhaustion genes (LAG3, TIGIT, TOX, HAVCR2) compared to the control group. In contrast, CD8+ T cells from controls showed upregulation of T cell activation and co-stimulation genes (ICOS, CD2, CD28, CD27). KEGG and Gene Ontology Term Biological Processes analysis reveals enrichment of pathways for ribosomal activity, cell proliferation, and negative regulation of T cell apoptosis in CD8+ T cells from controls, while CD8+ T cells from the CED group were enriched for pathways related to apoptotic signaling, negative regulation of T cell activation, and EBV infection. These data pinpoint specific functional immune alterations in the T cell compartment of individuals that are unable to mount effective responses to EBV.
11 150967 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #11 0b3b3924-8791-41ae-b8e5-bdab6009af25 No Thu, 09/04/2025 - 18:11 Thu, 09/04/2025 - 18:11 Thu, 09/04/2025 - 18:11 Anonymous 10.208.24.100 Rena R. Xian MD Associate Professor Johns Hopkins University School of Medicine Baltimore Baltimore rxian1@jhmi.edu EBV methylation, bisulfite sequencing, plasma, and saliva cfDNA, Wp Investigating the EBV methylome in PLWH: Discovery and Development of Novel EBV Diagnostics in Plasma and Saliva Introduction: EBV DNA derived from tumors is consistently methylated with unique methylation profiles. In contrast, virion DNA is never methylated. This project seeks to study the relationship between the EBV epigenome and EBV transcriptome in EBV(+) lymphoma in people with HIV (PWH). We will characterize EBV methylation in cell-free DNA (cfDNA) from plasma and saliva to define methylation patterns in different disease states in order to develop novel diagnostics for HIV-associated lymphoma.
Methods: EBV methylation was evaluated by a bead-based methyl-DNA binding protein capture of the W promoter and bisulfite sequencing of the viral genome (bsEBV-seq). Plasma samples from PWH with Hodgkin lymphoma (HL, n=12) or Plasmablastic lymphoma (PBL, n=21) were analyzed by the bead-based method. Thirty-two samples (tissue biopsy, plasma, and saliva) underwent bsEBV-seq. Lastly, cfDNA from 71 plasma samples and 44 matched saliva samples from PWH without EBV(+) lymphoma were evaluated for the presence of EBV.
Results: Bead-based methylation of Wp shows hypermethylation in HL (median 97%, 83-98%) and variable methylation in PBL (median 47%, 3-96%). bsEBV-seq shows diagnosis-specific and lymphoma subtype-specific patterns of methylation. HL shows consistent methylation across the EBV genome, whereas EBV methylation is more variable in PBL. Regarding OriP, there is either no methylation of the entire region, or there is recurrent site-specific hypermethylation. When evaluating EBV in PWH without EBV(+) lymphoma, the frequency of detecting EBV is higher in saliva (94%, 44/47) than in plasma (67%, 48/71). The level of EBV is also higher in saliva (median 3.8 LogEBVcopies/mL) than in plasma (0.8 LogEBVcopies/mL). bsEBV-seq of select saliva samples show no methylation across the viral genome consistent with presence of virion DNA.
Conclusions: Different methods of analyzing EBV methylation were applied. Findings from the EBV methylome studies will enable the development of novel plasma and saliva diagnostics for lymphoma in PWH.
4 150768 EBV-Associated Lymphoma Consortium Annual Meeting (Abstract): Submission #4 3c6f0dbc-efc3-4dbb-9ea0-27aaa8c50684 No Wed, 09/03/2025 - 13:22 Wed, 09/03/2025 - 13:22 Wed, 09/03/2025 - 13:22 Anonymous 10.208.28.30 Ben E. Gewurz MD, PhD Associate Professor Brigham & Women's Hospital Boston Boston bgewurz@bwh.harvard.edu EBV Latency Gene and Linker Histone H1 Cross-regulation in B cell lymphomagenesis Much remains to be learned about how EBV latency genes and host epigenetic factors cross-regulate one-another, with relevance to the development of novel host-targeted therapeutic approaches. While linker histone H1 are B-cell tumor suppressors mutated with striking frequency across EBV-negative lymphomas, little is known about histone H1 roles in EBV-mediated lymphomagenesis and lymphomas, how EBV latency genes suppress H1 expression or how H1 in turn cross-regulates the EBV epigenome. We build upon our observation that EBV strongly reduces linker histone H1 expression in newly infected B-cells as they transform into lymphoblastoid cells with the latency III program and in lymphoblastoid cell lines. We characterized how EBV suppresses histone H1 expression in B cell transformation. ATACseq highlighted that EBV latency III reduced H1 gene chromatin accessibility in Burkitt B cells, but not in newly transformed peripheral blood B cells or in LCLs. Histone H1 genes retained activating chromatin marks following B cell EBV infection and in LCLs. Instead, we observed reduction in histone H1 locus long-range chromatin contacts. While infection by EBNA3A/C double knockout (KO) EBV reduced H1 gene chromatin accessibility in LCLs, we observed increased H1 gene expression in B cells infected by EBNA3A KO EBV. However, EBV must maintain a some H1 expression to maintain viral latency. Relatedly, we investigated how H1 abundance correlated with B cell genome-wide H3K36me2 and H3K27me3 epigenetic marks, since the H3K36me2 epigenetic writers compete with H1 at DNA sites, whereas H1 promotes repressive H3K27me3 deposition to silence target genes. We used ChIP-seq to define target genes with the most differential H3K36me2 and H3K27me3 marks in latency I vs III states with elevated versus repressed H1 expression, respectively. Together, these studies provide key insights into how EBV latency genes and linker histone H1 cross-regulate one-another.