Mechanisms of epstein-barr virus (EBV) lytic cycle reactivation by a novel compound E11 intermediate (E11.1) in EBV-associated epithelial malignancies

Abstract

The degree of Epstein-Barr Virus (EBV) lytic cycle reactivation is one of the limiting factors in improving the efficacy of lytic induction therapy, an emerging therapeutic strategy against EBV-associated epithelial malignancies such as gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). The currently known classes of lytic inducers, for example, histone deacetylase (HDAC) inhibitors, have limited success in reactivating EBV lytic cycle in the different cellular background of GC and NPC. I endeavor to discover the novel modes of lytic induction of a new lytic inducing compound, known as E11.1, for use whether singly or in combination with others to improve the degree of lytic cycle reactivation. This study aims to investigate the biological effects of E11.1 on GC and NPC cells and delineate its distinct EBV lytic induction properties as compared to those of the other lytic inducing compounds. The mechanisms of action of E11.1 in reactivating EBV lytic cycle were examined by pharmacological inhibition and siRNA knockdown of several cellular pathways of interest. Different combinations of E11.1 and HDAC inhibitors were studied for their effects on lytic induction and cell death. E11.1 elicited EBV-specific inhibition effects on GC and NPC cells compared to their EBV-negative counterparts and potently induced EBV lytic cycle reactivation resulting in the generation of infectious viral particles in GC cells. Time-dependent cell cycle analysis on GC cells revealed that E11.1 readily induced an increase in the S-phase population at early timepoints, which provided favorable conditions for lytic induction, subsequently led to G2/M cell cycle arrest and caspase-dependent cell death at the later time points. E11.1 treatment was found to briskly activate protein kinase B (Akt), c-Jun N-terminal kinase (JNK), p38 mitogen-activated kinase (p38-MAPK) and MAPK/ERK kinase (MEK) signaling in GC and NPC cells. Applications of pathway inhibitors then narrowed down on the specific pathways being potentially involved in the lytic induction by E11.1. siRNA knockdown experiments further confirmed that p38α, JNK and c-Fos activation mediated E11.1’s reactivation of EBV lytic cycle in GC cells. Interestingly, the combination of E11.1 with the HDAC inhibitor, romidepsin, exerted a significant increase in the degree of lytic induction whilst that of E11.1 with SAHA did not augment the lytic induction. In conclusion, I have characterized the biological effects of E11.1 in terms of induction of EBV lytic cycle, cell cycle arrest and cell death in the EBV-associated epithelial cancers and uncovered that the mechanism of lytic cycle induction might be related to JNK-mediated or non-JNK-mediated activation of c-Fos. I have further demonstrated that combining E11.1 with romidepsin can potentiate the lytic induction of EBV in EBV-associated epithelial cancers.