Epigenetic Activation of the EBV Protein Kinase Determines Antiviral Drug Response in Central Nervous System Lymphoma

Epstein-Barr virus (EBV)-associated central nervous system lymphoproliferative diseases (CNSL) are aggressive clinical conditions with poor prognosis. We have reported that durable responses in patients with primary CNS post-transplant lymphoproliferative disease (PTLD) were associated with detection of two ganciclovir (GCV)/zidovudine (AZT) viral drug target proteins, the EBV kinases BGLF4 and BXLF1 in CNSL biopsies. These are associated with lytic EBV and the mechanism for expression in latently infected EBV+ CNSL has been unknown. By carrying out RNA expression analysis in CNSL tissue biopsies (n=12), we confirmed expression of LMP1, BXLF1, and BGLF4, but not BZLF1, pointing to an incomplete lytic EBV program. Biopsies from systemic PTLD (n=24) were used for comparison and showed significantly less expression of BGLF4. By quantifying DNA methylation in EBV gene promoters we showed significantly decreased promoter methylation at BGLF4 in CNSL versus systemic PTLD (p=0.0006). Luciferase reporter analysis of the BGLF4 upstream sequence revealed 3 regions of promoter activity and 5ˈ RACE in n=4 EBV-infected cell lines and n=5 CNSL biopsy samples identified transcription start sites at these promoters. We identified DNA methylation loss at single CpG dinucleotides which were specifically demethylated in CNSL, while surrounding EBV methylation remained high. Lastly, TET knockout and expression of TET1/2-suppressive mutant IDH1 in a latent HEK293 EBV model indicated that active demethylation is necessary for activity of BGLF4 promoters. We detail the epigenetic basis of BGLF4 expression in CNSL via locus-specific promoter activation that may hold value for determination of antiviral drug sensitivity.