IMMU-40. DECITABINE PRIMES GLIOBLASTOMA TO NY-ESO-1 SPECIFIC T-CELL CYTOLYSIS VIA DNA DEMETHYLATION OF NY-ESO-1, CANCER-TESTIS ANTIGENS, AND PRO-INFLAMMATORY SIGNATURES

Abstract

Abstract INTRODUCTION Previous work has shown that the DNA hypomethylating agent, Decitabine (DAC), can improve the immunogenicity of Glioblastoma (GBM) for immuno-therapeutic targeting through the upregulation of cancer testis antigens (CTA). Specifically, our group has published preclinical proof of principle that DAC upregulates NY-ESO-1, a well-studied and highly immunogenic CTA, in GBM for targeting by systemic adoptive T-cell therapy. However, the exact mechanisms by which this DAC-induced immunogenic enhancement occurs in GBM remains to be elucidated. We hypothesize that hypomethylating agents like DAC enable targeted immune therapy to overcome low immunogenicity in GBM cells via CpG demethylation of antigens and subsequent upregulation of inflammatory and antigen presentation gene signatures. METHODS We induced NY-ESO-1 expression in vitro in immortalized GBM lines and primary gliomaspheres using DAC and investigated the response of DAC-treatment using targeted bisulfite sequencing and quantitative real-time PCR. To functionally assess for specific immune killing of DAC treated GBM cells, we co-cultured DAC-treated cell lines with T-cells transduced to express a NY-ESO-1 specific T-cell receptor. Lastly, we performed probe-based methylation array and bulk RNA sequencing to interrogate global transcriptomic and epigenomic changes resulting from DAC treatment. RESULTS Sustained exposure to low dose (0.5 uM – 1 uM) DAC in immortalized and primary cell lines resulted in time-dependent demethylation of the CpG island of NY-ESO-1 and the subsequent robust increase in mRNA expression. Upregulation of NY-ESO-1 rendered specific T cell recognition and lysis of immortalized and primary cell lines. DAC upregulated other pro-inflammatory and immuno-modulatory signatures via CpG demethylation to enhance T-cell mediated targeted cytolysis of GBM. CONCLUSION Hypomethylating agents like DAC upregulate immunogenic signatures in primary and immortalized GBM cells, rendering them susceptible to antigen specific T-cell targeting. These pleiotropic effects demonstrate strong preclinical rationale for use of DAC to sensitize GBM for targeting by immune therapies.