111 Inhibition of EZH2 upregulates MHC I and II expression in oesophageal adenocarcinoma

Abstract Background Anti-programmed cell death protein1 (anti-PD-1) based immunotherapies improve survival in oesophageal adenocarcinoma (OAC) patients, but not all patients benefit. Inhibitors of the histone methyltransferase EZH2 (EZH2i) have been shown upregulate major histocompatibility complex (MHC) class I and II expression which overcame anti-PD-1 resistance in other cancers. This in vitro study assessed the efficacy of EZH2i and their impact on MHC expression in OAC. Methods Three OAC cell lines (FLO1, MFD1, OE33) and a EZH2i-sensitive line (HS-SY-II) were cultured in 2D and treated with a EZH2i panel. Cell viability (via EC50 calculation) was assessed via MTS assay. Differential gene expression analysis followed by gene set enrichment analysis of RNA-sequencing data from the cell lines treated with EZH2i or DMSO control was performed in R. Flow cytometry of MHC Class I (HLA-ABC) and II (HLA-DR) was performed in parallel replicate experiments. Results EZH2i demonstrated similar efficacy in all 4 cell lines (EC50 > 5 µM). In the RNA-sequencing data, EZH2i was associated with gene set enrichment of interferon alpha and gamma signalling pathways (p<0.001). In FLO1 cells, HLA-ABC stain intensity was significantly increased (p=0.006) following EZH2i but not the proportion of cells expressing HLA-ABC (99.3% vs 99.0%). EZH2i increased in the proportion of cells expressing HLA-DR (18.3% vs 1.0%, p=0.001). Conclusion Our data demonstrate the potential of EZH2i as a therapy to upregulate tumour cell MHC 1 and 2 expression which in turn may enhance anti-PD-1 immunotherapy response in OAC. Future work requires investigating EZH2i in combination with anti-PD-1 and chemotherapies.