Activation of Epstein–Barr Virus’ Lytic Cycle in Nasopharyngeal Carcinoma Cells by NEO212, a Conjugate of Perillyl Alcohol and Temozolomide

Simple Summary Nasopharyngeal carcinoma (NPC) is a cancer that is frequently associated with Epstein–Barr virus (EBV) infection, and this virus is thought to play a role in NPC development and malignant growth. Within tumor cells, the virus generally remains in a latent stage that supports cell survival and stimulates tumor growth. However, it has been postulated that it might be beneficial as part of cancer therapy to coax the virus out of its latent stage and into a lytic mode, which would result in tumor cell killing. A few agents are known to accomplish this switch, but their clinical success so far has been mixed. We are developing a novel compound, NEO212, that has shown anticancer activity in several preclinical tumor models. Here, in this report, we demonstrate that it is able to coax EBV into its lytic cycle, resulting in NPC cell death. We propose that NEO212 should be investigated further for its potential clinical usefulness as a therapeutic agent for NPC. Abstract The Epstein–Barr virus (EBV) is accepted as a primary risk factor for certain nasopharyngeal carcinoma (NPC) subtypes, where the virus persists in a latent stage which is thought to contribute to tumorigenesis. Current treatments are sub-optimal, and recurrence occurs in many cases. An alternative therapeutic concept is aimed at triggering the lytic cycle of EBV selectively in tumor cells as a means to add clinical benefit. While compounds able to stimulate the lytic cascade have been identified, their clinical application so far has been limited. We are developing a novel anticancer molecule, NEO212, that was generated by covalent conjugation of the alkylating agent temozolomide (TMZ) to the naturally occurring monoterpene perillyl alcohol (POH). In the current study, we investigated its potential to trigger the lytic cycle of EBV in NPC cells in vitro and in vivo. We used the established C666.1 cell line and primary patient cells derived from the brain metastasis of a patient with NPC, both of which harbored latent EBV. Upon treatment with NEO212, there was an increase in EBV proteins Zta and Ea-D, key markers of the lytic cycle, along with increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum (ER) stress, followed by the activation of caspases. These effects could also be confirmed in tumor tissue from mice implanted with C666.1 cells. Towards a mechanistic understanding of these events, we used siRNA-mediated knockdown of CHOP and inclusion of anti-oxidant compounds. Both approaches blocked lytic cycle induction by NEO212. Therefore, we established a sequence of events, where NEO212 caused reactive oxygen species (ROS) production, which triggered ER stress and elevated the levels of CHOP, which was required to stimulate the lytic cascade of EBV. Inclusion of the antiviral agent ganciclovir synergistically enhanced the cytotoxic impact of NEO212, pointing to a potential combination treatment for EBV-positive cancers which should be explored further. Overall, our study establishes NEO212 as a novel agent able to stimulate EBV’s lytic cycle in NPC tumors, with implications for other virus-associated cancers.