Glucocorticoid receptor suppresses GATA6-mediated RNA polymerase II pause release to modulate classical subtype identity in pancreatic cancer

Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a 5-year survival rate of 12%. It has two major molecular subtypes: classical and basal, regulated by the master transcription factors (MTFs) GATA6 and ΔNp63, respectively. Objective This study sought to uncover the transcriptional regulatory mechanisms controlling PDAC subtype identity. Design We integrated primary tumour single-cell RNA-seq, patient-derived xenograft RNA-seq and multispectral imaging to identify MTF-dependent, subtype-specific markers. We created subtype-specific fluorescent reporter systems and conducted drug screenings to find actionable targets. We analysed chromatin accessibility (ATAC-seq), genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac), MTFs (GATA6, ΔNp63), RNA polymerase II (Pol II), H3K4me3-anchored chromatin topology (HiChIP) and nascent RNA capture sequencing (PRO-seq). Additionally, we used nuclease-dead Cas9 (dCas9) to manipulate transcriptional regulatory mechanisms. Results Our approach identified glucocorticoid receptor (GR) agonists as agents that suppress the classical transcriptional programme by interacting with GATA6. GATA6 regulates classical-specific transcription through promoter-proximal pause release. Depletion of GATA6 increased Pol II occupancy at GATA6-bound enhancers and transcriptional start sites, stabilising enhancer–promoter interactions. Artificially inducing pausing at GATA6-bound enhancers with dCas9 abrogated target gene expression and induced pausing at both the enhancer and target gene promoter. Conversely, in basal PDAC ΔNp63 promotes Pol II recruitment and stabilises enhancer–promoter interactions. Conclusion This study provides new insights into the transcriptional control and role of GR agonists in controlling PDAC molecular subtype identity.