Novel small organic compounds for induction of Epstein-Barr virus (EBV) lytic cycle in EBV-positive epithelial malignancies

Abstract

Epstein-Barr virus (EBV) persists in its associated tumours in tight latent state, expressing few viral proteins. Induction of viral lytic cycle leads to the expression of a larger number of viral proteins which may paradoxically have detrimental effects to the tumour cells. Current main classes of EBV lytic cycle inducers are phorbol esters and histone deacetylase (HDAC) inhibitors. Both classes of chemicals mainly act through protein kinase C (PKC) pathway to induce lytic cycle. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may help to define mechanisms of lytic cycle activation in greater precision and increase the tumour cells’ responsiveness towards induction of lytic cycle.

We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. These compounds are structurally diverse themselves and distinct from phorbol esters or HDAC inhibitors. They neither caused hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, substantiating their difference with the known lytic inducers. Among the five compounds, one that could consistently induce complete lytic cycle progression (compound E11) and one that could induce lytic cycle in all the GC and NPC cell lines (compound C7) were selected for further study into their mechanisms of action.

Pharmacological inhibition of the phosphatidylinositol 3-kinase (PI3K), MAPK/Erk kinase (MEK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated kinase (p38 MAPK), protein kinase C δ (PKCδ) and ataxia telangiectasia mutated (ATM) did not significantly or consistently inhibit lytic cycle induction by the two selected compounds, suggesting that these pathways were not the main mediators of their lytic induction activity. Reactive oxygen species (ROS) scavengers consistently abolished EBV lytic cycle induction by compound E11, suggesting that ROS mediated the induction by E11 independent of the MAPKs. Compound C7 was noted to exhibit structural similarity to iron chelators and coordinate iron, copper and zinc ions with similar strength. It up-regulated the expression of the N-myc downstream-regulated gene 1 (NDRG1), a gene reported to be specifically regulated by intracellular iron level. Intracellular metal chelation was shown to be essential for compound C7’s lytic induction activity as pre-complexation of compound C7 to iron or zinc prior to treatment abolished its ability to reactivate lytic cycle. In parallel, several iron chelators of different structures were shown to induce EBV lytic cycle in EBV-positive carcinoma cells. Taken together, C7 acted as an iron chelator inside cells and induced lytic cycle by chelating intracellular metal ions.

In conclusion, we have identified two novel potent EBV lytic inducers in EBV-positive carcinoma that are structurally and biologically unrelated to the known lytic inducers. Their mechanism of action provided new insights into the regulation of EBV lytic reactivation.