Abstract B031: Loss of GATA6 leads to reduced cell fitness and PDA molecular subtype changes

Background: Pancreatic ductal adenocarcinoma (PDA) is defined by two main transcriptional subtypes, Basal-like & Classical. Classical tumors are more frequent and tend to respond better to chemotherapy with higher expression of genes related to pancreatic lineage development, the epithelial compartment, and adhesion. Conversely, Basal-like tumors are more aggressive, therapy resistant and have higher expression of laminins and keratins. The Classical subtype correlates with GATA6 expression, and ~16% pancreatic tumors show amplification in the transcription factor GATA6 suggesting its importance in the Classical subtype and in tumor survival (Chan-Seng-Yue et al., 2020). The goal of this project is to elucidate the role of GATA6 expression in PDA tumors showing that GATA6 is a key molecular regulator of the Classical subtype influencing tumor growth, maintenance, and phenotype. Methods: Patient derived organoids (PDOs) spanning the spectrum of GATA6 expression were used with CRISPR/Cas9 to establish GATA6 KO lines (n=8). These were then subject to a growth competition assay between cells with and without GATA6 KO and the amount of knockout over time was monitored by Sanger sequencing. Tumor growth curves were generated in a subcutaneous xenograft mouse model over 1.5 months to assess the in vivo growth potential of GATA6 KO cells in a sample with high GATA6 expression. 3’RNA-seq was used to assess gene expression changes in subtype, and GSEA was used to assess pathway enrichment. This was done alongside IHC, capillary Westerns, and flow cytometry to assess changes that GATA6 KO incurs on a functional level. Results: GATA6 KO tended to result in reduced fitness (dropout) in cells when ablated, although the rate and level of dropout varied, suggesting different levels of dependence on GATA6 expression for survival/growth. This was confirmed in vivo when the GATA6 KO failed to grow as robustly as control groups in a sample with high native GATA6 expression. Molecular analysis of all PDO samples showed shifts in expression from Classical to Basal-like. This shift was denoted in changes in subtype score, changes in marker gene expression (i.e., ANXA10 & TP63), and in changes in key pathways associated with each subtype. This shows that GATA6 not only correlates with the Classical subtype but plays a causal role in maintaining it. Even though there was a shift towards Basal-like expression several key characteristics of the subtype were notably absent including