Interrogating the functionality of accessible chromatin in Barrett’s oesophagus and oesophageal adenocarcinoma

Abstract

Oesophageal adenocarcinoma (OAC) is a major cause of global cancer-associated deaths. Despite the impact of this condition, we know relatively little about the mechanisms underlying the onset of this disease. OAC occurs from a pre-malignant lesion known as Barrett’s oesophagus (BO). There is a suggestion that alterations to the chromatin landscape may result in a transition to cancer. The functional significance of these changes is not understood, as of yet. To further our understanding of the molecular switch to disease, we have integrated total ribonucleic acid (RNA)-sequencing (seq) and assay for transposase accessible chromatin (ATAC)-seq data from BO and OAC patient tissue samples, and performed comparisons in order to identify state-specific enhancer RNAs (eRNAs). This approach uncovered almost 1000 OAC-specific eRNAs that are enriched in cancer-associated processes, including enhancers regulating the expression of junction plakoglobin (JUP), cyclin-E1 (CCNE1) and (Myb proto-oncogene like 2) MYBL2. Building on this work, we utilise the massively parallel reporter assay (MPRA) (self-transcribing active regulatory region) STARR-seq to functionally interrogate regions of chromatin associated with activity, marked by accessibility and the presence of marks such as Bromodomain-containing protein 4 (BRD4), H3K27ac and Med1. By integrating the STARR-seq assays into a high-confidence set, we identified 473 active enhancer elements in the OE19 OAC cell line. This candidate set is enriched for OAC-related processes. A subset of these high-confidence elements demonstrate a response to lapatinib, a small molecule inhibitor of the receptor tyrosine kinase Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), which is frequently amplified in OAC. Among the target genes of the lapatinib-sensitive enhancers is Pyruvate dehydrogenase kinase isoform 2 (PDK2), a gene which is involved in glycolysis, a process known to play a role in the resistance to chemotherapies in OAC. Thus, we demonstrate how enhancer-centric interrogation of the chromatin landscape can uncover biological understanding about the molecular mechanisms of disease, potentially paving the way for future therapies.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Magnus Rattray (Supervisor) & Andrew Sharrocks (Supervisor)