Abstract

Individuals with a functional T cell impairment, including the elderly, people living with human immunodeficiency virus (HIV), patients with inherited immune deficiencies, and those receiving iatrogenic immune suppression (organ transplant recipients or sufferers of autoimmune diseases) are at increased risk of developing aggressive lymphomas driven by the oncogenic Epstein-Barr virus (EBV). EBV is a ubiquitous herpes virus that infects >90% of adult humans. EBV can cause a self-limiting acute infectious mononucleosis, characterized by lytic viral replication, which is marked by the expression of lytic proteins such as BZLF1, and potent T cell-driven immune responses. EBV ultimately establishes a life-long latent persistence in the memory B cell compartment. The virus avoids complete eradication due to the ability of the latent infection to evade immune responses by limiting the expression of immunogenic viral proteins, inducing T cell senescence, Th1 cell differentiation blockade, and mobilization of regulatory T cells. Latent EBV drives lymphoproliferative disease (EBV LPD) development in the immunosuppressed setting by contributing to the immortalization of B cells. EBV LPD is frequently aggressive and has no unified standard-of-care treatment. Relapsed/refractory disease has a poor prognosis, and development of novel therapies is urgently needed. The expression of viral antigens in EBV(+) lymphoma cells makes immune-based therapies an attractive experimental option for treating relapsed/refractory EBV LPD. Restoration of EBV-specific T cell responses against BZLF1 is known to correlate with recovery in patients with EBV-driven post-transplant lymphoproliferative disease. Protein arginine methyltransferase 5 (PRMT5), a methyltransferase enzyme with driver activity in multiple hematologic and solid malignancies, is overexpressed in EBV LPD. PRT382 is a small molecule inhibitor of PRMT5 that has been successfully used in in-vitro and in-vivo models of hematologic malignancies. Our preliminary data showed that in vitro treatment with PRT382 arrests the growth of EBV-transformed lymphoblastoid B cell lines (EBV LCLs) derived from six healthy donors. PRT382 also enhanced gene transcription (up to 16X, p<0.05) and protein expression of the highly immunogenic lytic EBV protein BZLF1 in EBV LCLs in a dose-dependent manner. BZLF1 is known to drive EBV viral genome replication during the early steps of the lytic cycle. We observed a global loss of EBV genomic DNA methylation in EBV LCLs treated with increasing doses of PRT382 (p<0.0001) and a simultaneous increase in EBV genome copy number (up to 16X, p<0.05) per cell, suggesting that PRT382 may contribute to induction of the early steps of the EBV lytic cycle. We also observed increased transcription of EBV lytic genes BMRF1 and BALF4. The effects of PRMT5 inhibition on EBV gene transcription and protein levels appear to be dependent on Mek/Erk/c-Jun intracellular signaling within EBV LCLs. Furthermore, we observed enhanced susceptibility of EBV LCL targets treated with PRT382 to cytotoxic killing by EBV-specific T cells, suggesting that increased expression of EBV immunogenic epitopes such as BZLF1, driven by PRMT5 inhibition, may contribute to direct T cell-mediated cytotoxicity (reproduced with EBV LCLs derived from 3 donors). Finally, PRT382 significantly prolonged survival in NSG mice engrafted with EBV LCLs, while contributing to an increase in circulating EBV copy number (p<0.01), a decrease of EBV genome methylation (p<0.001) and increase in BZLF1 expression in the malignant EBV(+) B cells harbored by these mice. Studies of the combined therapeutic effects of PRMT5 inhibition and EBV-specific T cells in mouse models of EBV LPD are ongoing. Gene expression profiling is also currently underway to evaluate the impact of PRMT5 inhibition on EBV and human genes. Our preliminary data convincingly establishes PRMT5 inhibition as a novel and potent tool for induction of expression of highly immunogenic viral proteins, such as BZLF1, in a model of EBV LPD with latency III EBV infection, which is characteristic of EBV-driven lymphoma of immunosuppressed states. This enables us to generate a powerful platform for exploring enhanced tumor immunogenicity and for testing and comparison of various EBV-targeting cellular therapy products.