Identification of transcriptionally-active human papillomavirus integrants through nanopore sequencing reveals viable targets for gene therapy against cervical cancer.

ABSTRACT

Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case-by-case basis among related malignancies. Next-generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long-read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally-active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally-active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV-associated cancers.