Elucidation of interplay between Epstein-Barr virus and host factors in regulating epigenetics in nasopharyngeal carcinoma through multi-omics analysis

Abstract

Nasopharyngeal carcinoma (NPC) has the highest incidence in Southeast Asia but is rare worldwide. In the endemic areas, Epstein-Barr virus (EBV) is detected in more than 95% of NPC cases. EBV is proposed to be an oncovirus as it induces distinct epigenetic changes and dysregulates cancer-relevant pathways in a tissue-specific manner for tumor transformation and progression in multiple cancer types. Recent studies have shown that genomic aberrations precede and facilitate EBV-persistent infection in NPC, indicating that NPC pathogenesis is initiated or promoted by the interplay of genetic susceptibility, somatic genomic aberrations, and epigenetic alterations in the host cells and EBV. This study integrated (i) whole-genome bisulfite sequencing (WGBS) and matched whole-exome sequencing (WES) for NPC clinical specimens, (ii) assay for transposase-accessible chromatin using sequencing (ATAC-Seq), and RNA-Seq on EBV-positive (EBV+) and EBV-negative (EBV-) NPC cell lines, (iii) publicly available NPC single-cell RNA sequencing (scRNA-seq) to systematically profile the genomic and epigenomic changes including somatic mutations, DNA methylation, and chromatin accessibility and to elucidate the interplay between EBV and host factors for epigenetic dysregulation in NPC. WGBS and bisulfite pyrosequencing targeting LINE1 (a surrogate marker of global methylation level) were used to profile the global DNA methylation changes of 48 NPC tumor tissues. In addition to the globally hypermethylated EBV+ NPC (HyperNPC; 81.25% cases), a novel EBV+ NPC epigenomic subtype characterized by global hypomethylation (HypoNPC; 18.75% cases) was discovered. The matched WES data showed that HypoNPC was associated with the APOBEC mutation signature (COSMIC signature 2). The APOBEC/MBD4-mediated base-pair excision (BER) pathway plays an important role in DNA demethylation. This result suggested that APOBECs orchestrate genomic and epigenomic changes in a subset of EBV+ NPC patients. Regardless of the global methylation pattern, the CpG island methylator phenotype (CIMP) was consistently detected in both molecular subtypes by WGBS. However, EBV infection alone in the normal immortalized nasopharyngeal cell lines was insufficient to induce methylation changes observed in NPC, suggesting other key players contributing to CIMP were absent in the normal condition. The EBV-associated differentially methylated regions (EBV-DMRs) were identified by comparing the methylome data from EBV+ and EBV- gastric cancer samples and NPC cell lines. The study discovered that the EBV-DMRs were significantly enriched for the binding sites of a chromatin master regulator, CTCF. Importantly, CTCF hemizygous loss and reduction of protein level were frequently observed in NPC. These data collectively indicated that CTCF dysregulation is one of the host factors critical for EBV-DMRs in NPC. The ATAC-Seq data showed that EBV modulated chromatin accessibility, preferentially the binding sites of NF-kB, AP-1, and SOX family members. Gene ontology analysis indicated that the EBV-associated differentially accessible regions were relevant to developmental, differentiation, and immune pathways, which are important for virus survival in the host. In summary, this study reveals the unexpected heterogeneity of DNA methylation in EBV+ NPC, highlights the roles of APOBECs in NPC pathogenesis, and identifies CTCF dysregulation as an important host factor interplayed with EBV for epigenetic dysregulation promoting virus survival and conferring fitness to the NPC cells.