title: Development of therapeutic approaches to target Epstein Barr virus related disease
Creators: name: Armfield, Kate Frederique
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: EBV, PI3K, mRNA translation stress, EBNA1.

description: Epstein-Barr virus (EBV) has been associated with several lymphoid and epithelial malignancies, such as Burkitt’s Lymphoma and Nasopharyngeal Carcinoma, due to latent infection properties. The only EBV protein produced in all latency profiles of EBV is Epstein-Barr virus nuclear antigen 1 (EBNA1), a protein that forms a homodimer that is responsible for ensuring the replication and persistence of the EBV genome in cells to ensure a lifelong infection. EBNA1 has been explored as a driver for oncogenesis due to its ability to interact with host proteins and cellular promoters, whilst evading the immune system. The EBNA1 domain called the Glycine-Alanine repeat (GAr) causes translation suppression, resulting in the prevention of MHC class I EBNA1 antigen presentation. The GAr mediated self-regulatory properties of EBNA1 may be oncogenic; the full mechanism of which requires elucidation but appears to utilise a host stress response mechanism. This study focuses on revealing possible therapeutic agents for controlling EBV disease through the inhibition of EBNA1 expression or function. In order to inhibit EBNA1 dimerisation function, eight peptides, attached to cell penetrating peptides, were synthesised and designed to mimic the region of EBNA1 responsible for stabilising homodimers. Peptides were applied to human B cell lines to determine toxicity, specificity to EBNA1 cell survival, and the effect on dimerisation through an optimised dimerisation assay. The peptides did not affect EBNA1 specifically and no effect on cell viability or homodimer level was observed. Confocal microscopy revealed that the peptides were unable to enter the nucleus and instead aggregated, indicating they were unsuitable for use as a therapeutic agent. To investigate the GAr mediated self-inhibition mechanism, epithelial cell lines were transfected with GAr containing or lacking EBNA1 constructs, and cellular protein expression level evaluated. Pathway analysis and the evaluation of protein expression changes in latency I and III cell lines and Eμ-EBNA1 transgenic tumour cells revealed PI3K signalling components, C-MYC, E2F1, and MDM2 as candidate host proteins involved in this mechanism. Drug inhibition of proteins involved in these pathways and the effect on EBNA1 was analysed in human BL cell lines and Eμ-EBNA1 tumour cells in culture. C-MYC, PI3K, and MDM2 inhibition caused a reduction in EBNA1 expression, with PI3Kδ inhibition appearing to cause cell death in EBNA1 dependent cell lines. CAL 101, a PI3K p110δ inhibitor that is FDA approved, was then analysed for potential therapeutic properties in vivo using transplanted Eμ-EBNA1 tumour cells that were tagged with RFP to facilitate analysis. Whilst PI3K inhibition was successful in causing Eμ-EBNA1 tumour cell death in culture, this was not translated in vivo. Two in vivo studies, one that targeted dissemination of tumour cells and the other for treating established tumours, both resulted in progressed but variable tumour infiltration that did not differ significantly from the vehicle treated controls. The link between the proteins MDM2, E2F1, C-MYC and PI3K reveals new therapeutic routes to target EBNA1 function and suggests that MDM2 may be involved in the EBNA1 self-inhibition mechanism. In summary, these findings contribute to understanding the mRNA translational stress response caused by the GAr of EBNA1, which acts to increase proliferation signals in EBNA1 containing tumour cells.