Targeting lytic cycle with ciclopirox derivative in Epstein-Barr Virus+ gastric cancer

Epstein-Barr Virus (EBV) is a ubiquitous human herpesvirus that infects around 90% of the global adult population. Upon infection, the virus typically establishes latency within the host cell, expressing viral genes that promote tumorigenesis, inhibit apoptosis, and suppress immune recognition of infected cells. Induction therapy was introduced to counter these effects by driving the switch from latent to lytic phase, which is a process called lytic reactivation. This transition results in cell lysis with viral particle release and the expression of viral proteins that can be targeted by antiviral agents and the immune system. Here, we developed a novel in-silico drug prediction method to identify compounds capable of inducing the EBV lytic cycle in various EBV+ epithelial malignancies. In vitro treatment of top candidates has successfully triggered lytic reactivation on EBV+ gastric cancer cell lines, namely, SNU719 and AGSBX1. Among these candidates, Ciclopirox (CPX), a clinically used antimycotic agent that has a fungistatic and iron chelation capability, showed strong activity in inducing the EBV lytic cycle, inhibiting proliferation and survival of EBV+ gastric cancer cells by blocking the cell cycle. RNA sequencing revealed that CPX-mediated lytic induction acts primarily through the hypoxia pathway, specifically via hypoxia-inducible factor 1-alpha (HIF1alpha). CPX treatment increased HIF1alpha binding to chromatin regions associated with genes involved in gastric cancer proliferation and migration. Co-treatment with a HIF1alpha inhibitor abolished both the cytotoxic and lytic-inducing effects of CPX, confirming the pathway's central role. Despite its efficacy, a major limitation of CPX is its instability. During CPX administration, excess iron from both intracellular and extracellular environments will lead to degradation and inactivation of CPX. Co-treatment with equal molarity of ferric ion has completely abolished the lytic-inducing ability of CPX. To address this, we adopt the pro-drug strategy by adding a protective group on CPX, creating a protected derivative mCPX. mCPX has shown comparable activity in lytic induction, while significantly improved resistance toward iron-mediated inactivation compared to CPX or another published pro-drug fosciclopirox. Finally, in an SNU719 xenograft, mCPX demonstrated superior in vivo anti-tumor activity relative to CPX. Altogether, using in-sillico drug prediction platform, we have successfully repurposed CPX as a lytic-induction-based anti-tumor agent against EBV+ gastric cancer, and developed the pro-drug mCPX to enhance its in vivo stability.