The Role of histone modifying enzymes in oesophageal adenocarcinoma: targeting of enhancer of zeste homolog 2 (EZH2)

Abstract

Oesophageal adenocarcinoma (OAC) is a cancer of unmet clinical need, with 5-year survival rates <20%. Owing to the high prevalence of loss of function mutations in the SWI/SNF chromatin remodelling subunit ARID1A, OAC tumours have a hypothesised sensitivity to inhibitors of the histone lysine methyltransferase EZH2 (EZH2i), based on observations from other ARID1A mutant cancers. This has not previously been explored in OAC.

Initially, through a bioinformatic analysis of a large OAC mRNA dataset (TCGA), high EZH2 mRNA expression was found to be independently associated with worse overall survival (OS). These findings were then validated using immunohistochemistry of a clinically annotated OAC tissue microarray series stained for EZH2 and associated histone 3 lysine 27 trimethylation mark (H3K27me3) which demonstrated EZH2 overexpression in OAC samples, but this was not prognostically significant.

The efficacy of EZH2i in OAC was then investigated using a panel of EZH2is alone or in combination with standard of care chemotherapies in cultured OAC cell lines and an EZH2i-sensitive synovial sarcoma cell line. EZH2i showed similar efficacy across all the cell lines and demonstrated a synergistic relationship with 5-FU/ docetaxel in one OAC cell line. Following generation of an ARID1A CRISPR knockout (KO) OAC cell line, EZH2i efficacy was shown to be reduced in the KO cell line compared to wild-type. In addition, ARID1A KO appeared to promote epithelial-mesenchymal transition of the OAC cells, with a morphological change to a more mesenchymal phenotype and associated upregulation of mesenchymal markers determined via western blot and immunofluorescence.

Finally, differential gene expression analysis followed by gene set enrichment analysis of mRNA sequencing data from EZH2i treated OAC cells demonstrated enrichment in gene sets involved in interferon alpha and gamma signalling pathways. There was an increase in OAC cell expression of major histocompatibility complex class I and II molecules determined by flow cytometry.

In conclusion, this study demonstrates for the first time the efficacy of EZH2i in OAC in vitro. Further, it shows a role for EZH2i in upregulating MHC class I and II expression, which may have clinical utility in OAC patients to improve response to immunotherapy treatment.

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Identifiers

ORCID for Oliver John Pickering: orcid.org/0000-0003-4100-385X

ORCID for Tim Underwood: orcid.org/0000-0001-9455-2188

ORCID for Zoë Walters: orcid.org/0000-0002-1835-5868

ORCID for Matthew Rose-Zerilli: orcid.org/0000-0002-1064-5350

ORCID for Jane Gibson: orcid.org/0000-0002-0973-8285

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