EBNA1 inhibitors reveal CDC7 and POU2F1 as direct functional targets in EBV epithelial cancers

ABSTRACT Epstein-Barr virus (EBV) latent infection is causally linked to several epithelial cancers, including endemic forms of undifferentiated nasopharyngeal carcinoma (NPC), and to a subtype of gastric cancer (GC). EBNA1 is the virus-encoded sequence-specific DNA-binding protein required for episome maintenance but also contributes to host-cell survival through multiple mechanisms, including binding to the host chromosome. We previously developed small-molecule inhibitors of EBNA1 DNA-binding that block host cell cycle progression and growth of EBV+ cell lines and tumor models in vivo . However, the underlying molecular mechanisms of EBNA1 function and inhibition have not been completely elucidated. In this study, we employ VK1727 to inhibit EBNA1 DNA-binding to viral and cellular genomes in three EBV+ epithelial tumor-derived cell models (patient-derived xenograft C15, C666-1, and SNU719). We integrate EBNA1 ChIP-seq and transcriptomic RNA-seq analyses to identify the cell cycle-dependent kinase CDC7 and a stem cell transcription factor POU2F1 as direct functional targets of EBNA1 in each of these epithelial cancer models. EBNA1 binding to the CDC7 promoter and POU2F1 intron promotes RNA Pol II-pS5 to initiate transcription of these two genes. We show that CDC7 inhibitor simurosertib phenocopies VK1727, while Bcl-2 inhibitor venetoclax shows a synergistic effect with VK1727 for inhibition of EBV+ epithelial cancer cell proliferation and survival. Our study reveals new functional gene targets and pathways of VK1727 in EBV+ epithelial cancers that provide new biomarkers and combinatorial strategies to treat EBV-driven cancers. IMPORTANCE EBNA1 is essential for Epstein-Barr virus (EBV) latency and tumorigenesis, but its mechanism of action on host gene expression is not yet known. Small-molecule inhibitors of EBNA1 DNA-binding block cell cycle progression and inhibit the growth of EBV+ tumors. In this study, we use the EBNA1 small-molecule inhibitor VK1727 to identify cellular gene targets that are bound by EBNA1 and deregulated by its pharmacological inhibition in EBV+ epithelial cancer cell lines and an NPC patient-derived xenograft mouse model. We identify cell cycle-dependent kinase CDC7 and the stem cell transcription factor POU2F1 as EBNA1-bound and regulated genes important for EBV epithelial cancer proliferation. These findings not only decipher the molecular mechanism by which VK1727 blocks cell cycle progression and inhibits cell proliferation but also provide two new cellular gene targets and pathways for therapeutic intervention in EBV+ epithelial cancers.