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Structure-based design of small-molecule inhibitors of EBNA1 DNA binding blocks Epstein-Barr virus latent infection and tumor growth.

ABSTRACT: Epstein-Barr virus (EBV) is a DNA tumor virus responsible for 1 to 2% of human cancers including subtypes of Burkitt's lymphoma, Hodgkin's lymphoma, gastric carcinoma, and nasopharyngeal carcinoma (NPC). Persistent latent infection drives EBV-associated tumorigenesis. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated tumors and is therefore an attractive target for therapeutic intervention. It is a multifunctional DNA binding protein critical for viral replication, genome maintenance, viral gene expression, and host cell survival. Using a fragment-based approach and x-ray crystallography, we identify a 2,3-disubstituted benzoic acid series that selectively inhibits the DNA binding activity of EBNA1. We characterize these inhibitors biochemically and in cell-based assays, including chromatin immunoprecipitation and DNA replication assays. In addition, we demonstrate the potency of EBNA1 inhibitors to suppress tumor growth in several EBV-dependent xenograft models, including patient-derived xenografts for NPC. These inhibitors selectively block EBV gene transcription and alter the cellular transforming growth factor-? (TGF-?) signaling pathway in NPC tumor xenografts. These EBNA1-specific inhibitors show favorable pharmacological properties and have the potential to be further developed for the treatment of EBV-associated malignancies.

SUBMITTER: Messick TE

PROVIDER: S-EPMC6936217 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Project description:The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.

Project description:As the second leading cause of death in the United States, cancer has a considerable impact on society, and one cellular process that is commonly dysregulated in many cancers is the post-translational modification of proteins by the Small Ubiquitin-like Modifier (SUMO; sumoylation). We documented that sumoylation processes are up-regulated in lymphoma tissues in the presence of Latent Membrane Protein-1 (LMP1), the principal oncoprotein of Epstein-Barr virus (EBV). LMP1-mediated dysregulation of cellular sumoylation processes contributes to oncogenesis, modulates innate immune responses, and aids the maintenance of viral latency. Manipulation of protein sumoylation has been proposed for anti-cancer and anti-viral therapies; however, known inhibitors of sumoylation do not only target sumoylation processes. Recently, a specific and selective small-molecule inhibitor of sumoylation (ML-792) was identified; however, nothing is known about the effect of ML-792 on LMP1-mediated dysregulation of cellular sumoylation or the EBV life-cycle. We hypothesized that ML-792 modulates viral replication and the oncogenic potential of EBV LMP1 by inhibiting protein sumoylation. Results showed that ML-792 inhibited sumoylation processes in multiple EBV-positive B cell lines and EBV-positive nasopharyngeal carcinoma cell lines but not in their EBV-negative counterparts. Focusing on its effect on B cells, ML-792 inhibited B-cell growth and promoted cell death at very low doses. ML-792 also modulated LMP1-induced cell migration and cell adhesion, which suggests the abrogation of the oncogenic potential of LMP1. Finally, while higher concentrations of ML-792 were sufficient to induce low levels EBV spontaneous reactivation, they decreased the production of new infectious virus following an induced reactivation and the infection of new cells, suggesting that ML-792 has anti-viral potential. Together, these findings suggest that ML-792 may be a potential therapeutic drug to treat EBV-associated lymphoid malignancies by targeting oncogenesis and the EBV life-cycle.

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Structure-based design of small-molecule inhibitors of EBNA1 DNA binding blocks Epstein-Barr virus latent infection and tumor growth.

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