Identifying Endogenous Retroviral Factors in Viral Lymphomagenesis

PROJECT SUMMARY/ABSTRACT Malignancy is the leading cause of mortality amongst people living with HIV (PLWH). Of these malignancies, Non-Hodgkin lymphoma (NHL) is the most common hematological neoplasm affecting PLWH. Despite the introduction of highly active antiretroviral therapy (ART), PLWH are 10-20 times more likely to develop NHL than those without HIV. Epstein Barr Virus (EBV) is an oncogenic virus that is associated with the lymphomas most commonly observed in PLWH. Active HIV infection can lead to Acquired Immunodeficiency Syndrome (AIDS) in untreated people, and it is known that AIDS promotes the development of EBV+ lymphomas, termed AIDS- defining cancers (ADCs), the effect of host factors, including endogenous retroviruses on lymphomagenesis in immunocompetent PLWH treated with ART, remains to be explored. Work in the Cesarman and Nixon labs has shown that EBV and HIV each hijack host cell signaling pathways upon infection to induce the expression of endogenous retroelements, including as human endogenous retroviruses (HERV). To determine the contributions of these viruses on lymphomagenesis, we will generate data from in vitro lymphoma models to test the effect of EBV and HIV proteins on the expression of endogenous retroviral oncoproteins. Additionally, we will perform bulk and single-cell sequencing of Diffuse Large B Cell Lymphoma (DLBCL) from PLWH to determine the changes in cell populations of the tumor microenvironment (TME). We hypothesize that there will be unique populations of EBV infected B cells with pre-malignant transcriptomic signatures that correlate with dysregulated HERV expression, including expression of the HERVK(HML2) Np9 oncogene. In Aim 1, we will determine the impact of EBV latency III LMP2A and extracellular HIV p17 variants on the expression of the Np9 oncogene. We will perform stepwise molecular and cellular studies to determine the translational impact of exogenous viral infection on the expression of this endogenous retroviral oncogene. We will additionally perform multi-omic analysis to identify viral and host factors relevant to the transcriptional machinery of virus-mediated Np9 oncogene expression. In Aim 2, we will use our novel established single cell pipeline to determine locus-specific endogenous retroviral transcription of DLBCL samples from PLWH. Preliminary data from our work in the Nixon lab strongly suggests that we will be able to use the pipeline that we have developed to identify and quantify these HERV transcripts from RNA-sequencing data. We will assess the relative cell populations of samples that are EBV+ to those that are EBV- and determine the differences in host gene, retroviral and viral transcripts. Additionally, we intend to validate these single cell findings with bulk RNA-sequencing from a larger cohort of EBV+/- fresh frozen tissue of DLBCL from PLWH.