Cancer has historically been considered a genetic disease originating from activating mutations of oncogenes or inactivating mutations of tumor suppressors. More recently, appreciation for the contributions of epigenetic mechanisms to carcinogenesis has grown as they help to explain the aberrant gene expression and cellular identity associated with many cancers. For example, changes in DNA methylation at CpG dinucleotides can alter gene expression to promote malignant properties. In pancreatic ductal adenocarcinoma (PDA), the genetic underpinnings of tumorigenesis are known and primarily originate from mutations in KRAS and TP53. Furthermore, transcriptomic studies have identified transcriptional programs that are commonly enriched in subsets of patients, resulting in two main subtypes of PDA with clinical implications: squamous-PDA and progenitor-PDA. An understanding of the genome-wide DNA methylation aberrations in PDA and their contribution to tumor progression and subtype identity is less clear. In this dissertation, the genome-wide DNA methylation changes across PDA stages and subtypes as well as the impacts on metastatic characteristics and subtype differentiation are explored. Chapter 1 is a review of the literature highlighting our current understanding of the DNA methylation landscape in PDA which has primarily been learned from microarray studies that assay a very limited subset of CpGs. In Chapter 2, we characterize the genome-wide DNA methylation landscape of PDA organoids using whole genome bisulfite sequencing (WGBS), identifying and characterizing stage-specific and subtype-specific DNA methylation differences that may be beneficial as prognostic and predictive biomarkers. In Chapter 3, we identify subtype-specific DNA methylation differences in commercially available PDA cell lines and dissect GATA6-mediated epigenetic contributions to progenitor-PDA subtype differentiation. GATA6 is a known master regulator of progenitor-PDA and we found that its binding motifs are enriched in hypermethylated regions of squamous-PDA. While restoration of GATA6 expression in squamous-PDA did upregulate the progenitor-PDA gene signature, it did not reverse the observed hypermethylation suggesting that GATA6 does not directly mediate DNA methylation of its binding regions. Instead, GATA6 may epigenetically regulate transcription of its target genes through its interaction with the SWI/SNF family of chromatin remodelers. Thus, the SWI/SNF complex may be a therapeutically actionable target for treating patients with the progenitor-PDA subtype. In Chapter 4, we perform a combined transcriptomic and DNA methylome analysis of mouse PDA organoid models to identify genes whose methylation-mediated silencing likely contributes to PDA metastasis. We found that hypermethylation of the Pdzrn3 promoter represses Pdzrn3 transcription and promotes metastatic characteristics including increased cell migration and a pro-migratory cell morphology. Chapter 5 summarizes the main findings of this work, applications of the research, and future directions.

Pancreatic ductal adenocarcinoma (PDA) presents a significant challenge in oncology due to its aggressive nature and limited treatment options. Despite advancements in understanding the genomic landscape of PDA initiation and progression, the mechanisms driving metastasis remain elusive. This doctoral thesis explores the critical role of epigenetic mechanisms, specifically focusing on transcriptional regulation, histone modification, and molecular crowding in PDA metastasis. Through a multidisciplinary approach, encompassing genomic analyses, functional studies, and biophysical investigations, the thesis sheds light on the intricate interplay between genetic and epigenetic factors driving PDA progression. Chapters elucidate the aberrant expression of the homeodomain transcription factor EN1 in PDA, its impact on downstream signaling pathways, and its promotion of PDA transformation and metastasis. Furthermore, the thesis uncovers the influence of nucleoplasmic macromolecular crowding, particularly highlighting the role of nesprin-3, in regulating nuclear dynamics and metastatic potential in PDA. By integrating genetic, epigenetic, and biophysical perspectives, this work not only advances our understanding of PDA pathogenesis but also paves the way for the development of targeted therapeutic interventions. Additionally, the thesis underscores the significance of pancreatic cancer organoid models in recapitulating disease progression, elucidating molecular dynamics, and guiding precision medicine approaches. Overall, this research underscores the importance of a comprehensive approach in deciphering the complexities of PDA and offers valuable insights for future therapeutic strategies aimed at improving patient outcomes.