ECOA-2. Immuno-sensitization of Glioblastoma to NY-ESO-1 Targeting via Promoter Demethylation

Abstract Introduction The immunotherapeutic targeting of New York-esophageal squamous cell carcinoma (NY-ESO-1) and other cancer/testis antigens (CTA) is an appealing strategy for the treatment of malignant gliomas because CTA are not expressed in most normal adult tissues and their expression can be induced in tumors for targeting by T-cells. Basally, NY-ESO-1 is often poorly expressed in glioblastoma (GBM), presumably through promoter methylation. Mechanisms governing the expression of CTA have been explored in other cancers; however, neither the prevalence of NY-ESO-1 downregulation in GBM patient tumors nor the presumed mechanism of downregulation by promoter methylation in GBM has been formally established. Methods We characterized baseline CpG methylation of NY-ESO-1 in 30 bulk patient GBM samples, 10 patient-derived gliomaspheres, and three established tumor cell lines using bisulfite sequencing. We induced NY-ESO-1 expression in vitro in U251 human GBM cells using the hypomethylating agent decitabine (DAC). We investigated the epigenetic response of DAC-treated U251 with bisulfite sequencing and NY-ESO-1 expression with quantitative real-time PCR. Lastly, we performed single-cell RNA sequencing on DAC-treated GBM U251 to evaluate tumor subpopulations that upregulate NY-ESO-1 and other co-expressed CTA after DAC treatment. Results Baseline NY-ESO-1 expression is associated with promoter methylation in the majority of GBM. Treatment of cells with 1 µM DAC every day for 4 days explicitly demethylated the promoter region of NY-ESO-1 and resulted in a 1000-fold increase in mRNA expression. DAC treatment upregulates NY-ESO-1 coordinately with other cancer/testis antigens CTAG2 and MAGEA4 as demonstrated by single-cell RNA sequencing. Conclusion Exposure of U251 to DAC results in promoter demethylation in NY-ESO-1 and increased expression of CTA. DAC treatment may therefore render GBM susceptible to targeting of these antigens by T-cells, revealing a feasible strategy of NY-ESO-1 and co-expressed CTA promoter demethylation to sensitize GBM to immunotherapy.