Cellular Mechanism of Reactivation of Lytic Cycle of Epstein-Barr Virus (EBV) by a Novel Compound, C7, in EBV-associated Epithelial Malignancies

Pharmaceutical reactivation of lytic cycle of Epstein–Barr virus (EBV) represents a potential therapeutic strategy against EBV-associated epithelial malignancies, e.g., gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). A novel lytic-inducing compound, C7, which exhibits structural similarity to Di-2-Pyridyl Ketone 4, 4-Dimethyl-3-Thiosemicarbazone (Dp44mT), a known chelator of intracellular iron, is found to reactivate EBV lytic cycle in GC and NPC. This study aims to delineate the mode of action of C7 and clinically available iron chelators in EBV lytic reactivation in GC and NPC and to determine the effect of combining C7 with ganciclovir (GCV) and with lytic inducers of EBV that possesses distinct mechanism in EBV lytic reactivation, i.e. histone deacetylase inhibitor (HDACi), in EBV lytic reactivation and the killing of EBV-positive cells. Previous study showed the activation of the hypoxia signaling pathway upon C7 or iron chelator treatment, however, in this study, it was verified that the hypoxia signaling pathway was not the only pathway associated with EBV lytic reactivation. Specifically, C7 initiated autophagy by activating extracellular signal-regulated kinase (ERK1/2) to reactivate EBV lytic cycle since autophagy and EBV lytic reactivation were abolished in cells treated with ERK1/2 blockers whilst inhibition of autophagy by 3-Methyladenine (3-MA) and atg5knockdown significantly abolished EBV lytic reactivation. Continuing with the previous finding, it was found that inhibition of autophagy initiation by 3-MA and autolysosome formation by chloroquine demonstrated that only autophagy initiation is required for EBV lytic reactivation. Gene knockdown of various autophagic proteins such as beclin-1, ATG3, ATG5, ATG7, LC3B, ATG10, AT12 and Rab9, revealed that only the knockdown of ATG5 diminished EBV lytic reactivation. 3-MA could only abrogate lytic cycle induction by C7 and iron chelators but not by HDACi, In addition, these two subclasses of lytic inducers impose different cellular effects and lead to distinct stages of cell cycle arrest in NPC cells. In conclusion, this study has introduced a new class of compounds, C7 and clinically available iron chelators and has characterized their mode of action in EBV lytic reactivation which is through intracellular iron chelation and the activation of the ERK1/2-autophagy (ATG5 in particular) axis, which is different from conventional lytic inducers, such as SAHA. This provides evidence to support the introduction of C7 and iron chelators to the conventional drug reservoir to enhance the efficacy as well as explore possible drug combination in the lytic induction therapy and to achieve specific killing on EBV-positive malignancies. Moreover, the discovery on the role of autophagy in EBV lytic reactivation enhanced our understanding of virus-host modulation of the EBV lytic cycle which could also open up potential novel strategies against EBV-associated cancers.