Investigating the transcription factor landscape of the squamous subtype of pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, characterised by late diagnosis and early onset of metastases, and has a dismal 5-year survival of only 8%. Based on gene expression profiles PDAC can be stratified into two distinct molecular subtypes: classical and squamous, with the latter being associated with poorer clinical outcome. The Carroll Lab is interested in the role of pioneer transcription factors and their chromatin associated interaction partners in regulating transcriptional programs in different cancer types. This thesis has a particular focus on discovering the transcription factors involved in the lesser-known squamous subtype of PDAC, and understanding how they regulate cis-regulatory regions and enhancer landscapes to maintain the PDAC squamous subtype. A proteomics approach developed within the lab called RIME (Rapid Immunoprecipitation and Mass spectrometry of Endogenous proteins) was used to identify the interactome of TP63 with an outlook to delineate its role in a subtype specific manner, and also identify potential downstream therapeutic targets. ChIP-seq and other genomics-based methods were used to map TP63 chromatin binding, and understand how the enhancer landscapes are affected when it is down regulated. The well characterised pioneer factor FOXA1 was identified as a top interactor of TP63, which were further shown to co-bind at transcriptional start sites and cis-regulatory elements across the genome in a panel of human squamous PDAC cell lines. Unexpectedly GATA6 which has historically been associated with being downregulated in this PDAC subtype is also found as a TP63 interactor, and shares many TP63 and FOXA1 common ChIP binding sites. Ongoing work aims to understand the interplay between TP63, FOXA1, and GATA6 and identify which of these is required for transcription factor recruitment, as well as regulating distal enhancer regions. Studying the TP63 and FOXA1 interactomes also identified several chromatin regulators which could potentially be used as novel therapeutic targets. Protein Arginine Methyl transferase 5 (PRMT5) shows particular promise as a target, with a panel of squamous cell line models showing significant sensitivity to PRMT5 inhibition in cell-viability and colony formation assays. As the squamous subtype of PDAC is very understudied, this work revealed the transcription factor landscape of this subtype, giving insight to how gene expression is regulated. This can also be compared to the classical subtype to understand how these subtypes differ from each other, and whether there are subtype specific targets which could allow for a more targeted approach to treating these tumours.