USP7 Inhibitors Destabilize EBNA1 and Suppress Epstein-Barr Virus Tumorigenesis

Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt's lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels in a proteosome-dependent manner. Proteomic analysis revealed that USP7 inhibitor GNE6776 altered the EBNA1 protein interactome, including disrupting USP7 association with EBNA1. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. Transcriptomic studies revealed that USP7 inhibition affected chromosome segregation and mitotic cell division pathways in EBV⁺ cells. Finally, we show that GNE6776 selectively inhibited EBV⁺ gastric and lymphoid cell proliferation in cell culture and slowed EBV⁺ tumor growth in mouse xenograft models. These findings suggest that USP7 inhibitors perturb EBNA1 stability and function and may be exploited to treat EBV latent infection and tumorigenesis.