Single-Cell Profiling of HDAC Inhibitor-Induced EBV Lytic Heterogeneity Defines Abortive and Refractory States in B Lymphoblasts

ABSTRACT Epstein-Barr virus (EBV) is associated with multiple malignancies including Burkitt lymphoma (BL), Hodgkin’s lymphomas, nasopharyngeal carcinomas (NPC), and gastric cancers. Canonically, EBV positive tumors display latent gene expression programs that are difficult to target pharmacologically. To overcome this hurdle, lytic reactivation therapies have been developed based on HDAC inhibition with limited mechanistic studies. We therefore characterized the impact of pan-HDAC inhibitor, panobinostat, and class I HDAC inhibitor, nanatinostat, on the growth, survival, and lytic reactivation of four EBV-positive cell lines: P3HR1-ZHT BL, Jijoye BL, IBL-1 immunoblastic lymphoma, and de novo infection derived lymphoblastoid cell lines (LCL). All lines were sensitive, enabling us to define ranges of sensitivity within which to use single cell approaches to assess early EBV lytic gene expression, cell cycle state, and apoptosis. We observed that each EBV-positive model of malignancy responded uniquely to the same HDAC inhibitors and that lytic reactivation was successful in only a small percentage of the cell population. To elucidate the potential role of host factors in preventing successful lytic reactivation, we performed single-cell RNA sequencing on the P3HR1-ZHT BL line treated with the HDAC inhibitor panobinostat. We observed that abortive lytic cells, or cells that do not successfully progress through the lytic cycle, upregulated genes downstream of NF-κB activity. Additionally, genes involved in immune signaling including the CD137/CD137L signaling axis, were upregulated in abortive lytic cells. These data have important implications for how we approach oncolytic therapies for EBV-associated malignancies. AUTHOR SUMMARY Epstein-Barr virus (EBV) is an extremely prevalent human herpesvirus that is associated with a variety of cancers and autoimmune diseases. EBV establishes latent infection in the host and, under various circumstances, can reactivate the lytic cycle to produce more infectious particles. In the context of EBV-associated malignancies, the virus is most often maintained in a latent state, which makes it difficult to target with pharmaceuticals. To develop more viral targeted strategies, kick and kill regiments have been investigated. This therapy involves reactivating the virus with an HDAC inhibitor followed by treatment with an antiviral drug. It is well established that reactivating EBV with pharmaceuticals is often inefficient and leads to heterogeneous responses, including an abortive lytic trajectory. To better characterize the overall effect of two classes of HDAC inhibitors in various models of EBV-associated malignancies, we utilized single-cell techniques to capture various responses to stimuli. Consistent with prior studies, HDAC inhibition led to both successful and abortive lytic populations. Single-cell RNA sequencing provided evidence of upregulated immune signaling pathways in this abortive lytic population. This study provides in depth characterization of lytic reactivation with a biologically relevant stimulus. Competing Interest Statement The authors have declared no competing interest.