Glucocorticoid Receptor Suppresses GATA6-Mediated RNA Polymerase II Pause Release to Modulate Classical Subtype Identity in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a five-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. This study aims to uncover the transcriptional regulatory mechanisms controlling PDAC subtype identity. We integrated primary tumor 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 analyzed genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac), MTFs (GATA6, ΔNp63), RNA polymerase II (Pol II), nascent RNA capture sequencing (PRO-seq), and H3K4me3-anchored chromatin topology (HiChIP). Additionally, we used nuclease-dead Cas9 (dCas9) to manipulate transcriptional regulatory mechanisms. Our approach identified glucocorticoid receptor (GR) agonists as agents that suppress the classical transcriptional program by modulating GATA6 activity. GATA6 regulates classical-specific transcription through promoter-proximal pause (PPP) release. Depletion of GATA6 increased Pol II occupancy at GATA6-bound enhancers and transcriptional start sites, stabilizing 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, ΔNp63 in basal PDAC promotes Pol II recruitment and stabilizes enhancer-promoter interactions. This study provides new insights into the transcriptional control of PDAC subtypes and suggests potential therapeutic strategies, highlighting the role of glucocorticoids in PDAC chemotherapy. To investigate the role of glucocorticoid receptor (GR) agonists and master transcription factors (MTF) GATA6 and deltaNp63 on subtype identity, we performed ChIPseq in classical cell lines AsPC1, HPAFII, PaTu8988s and TCCPAN2 and classical PDX models GöPDX5 and GöPDX8 and basal cell line L3.6pl and T3M4 under control conditions or after GR agonist treatment, siRNA depletion of MTFs in respective cell lines, or CDK9 inhibition with Enitociclib.