Abstract C071: Transcription factor switching drives progression of the classical subtype of pancreatic cancer

Abstract Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late-stage disease and are often unable to tolerate drug combination regimens. Large-scale genomic approaches categorise PDAC into two distinct molecular subtypes, based on transcriptomic signatures, and these are termed the classical and squamous (or basal) subtypes. Although it is well-described that PDAC harbours gene mutations (i.e. KRAS G12D, p53 R172H) that initiate the disease, little is known about the influence of lineage-defining Transcription Factors (TF) on PDAC subtypes and their contribution to tumor progression. We took an unbiased approach to discover driving TFs in PDAC, we performed ChIP-seq of the active enhancer mark H3K27Ac from surplus tissue following resection of primary PDAC tumors (n=6) and normal adjacent tissue (n=4). Analysis of the H3K27Ac sites showed that the most enriched motifs within all experimentally mapped enhancer elements were HNF motifs (HNF4G & HNF4A) and Forkhead (i.e., FOXA1), confirming that these two classes of TFs constitute the lineage-defining factors in these clinical samples. Investigation of publicly available clinical datasets revealed HNF4G to be amplified in 9% of PDAC patients. Using Rapid Immunoprecipitation and Mass Spectrometry of Endogenous protein complexes (RIME), our method for unbiased discovery of endogenous protein complexes, we investigated the fundamental differences in the FOXA1 interactome between models of classical and squamous subtypes. HNF4A and HNF4G were only seen as FOXA1-interactors in models of the classical subtype. HNF4G Knockout (KO) clones injected orthotopically into the pancreas of mice, showed a significant increase (p-value<0.0006) in median survival and slower tumor growth compared to Control (Cas-9) (p-value <0.0001). HNF4G-KO dependent genes were specifically expressed in primary tumors (with decreased EMT signalling) versus metastatic samples (PanCRux dataset), implying that the HNF4G target genes were mostly limited to primary tumor contexts. We found Protein Arginine Methylate Transferase (PRMT1) to be a significant and reproducible interactor of FOXA1 and HNF4G. We observed a significant global reduction in PRMT1 chromatin binding in both the HNF4G-KO and FOXA1 depleted cells compared to the control. This data suggests a dependency on both HNF4G and FOXA1 for recruitment of PRMT1 to chromatin. We find HNF4G to be the key driver of classical subtype primary disease, in part by recruiting the methyltransferase PRMT1 to regulatory regions revealing a therapeutically exploitable opportunity. A molecular switch occurs in advanced disease, whereby HNF4G expression decreases, unmasking FOXA1’s transcriptional activity. De-repressed FOXA1 drives late-stage disease, by orchestrating metastasis-specific enhancer-promoter loops to direct metastatic gene regulation. We suggest the existence of stage-dependent TF activity, triggered by molecular compartmentalisation that mediates multistage progression of PDAC. Citation Format: Shalini Vasudev Rao, Lisa V Young, Danya Cheeseman, Stephanie V Mack, Dominique-Laurent Couturier, Sean V Flynn, Rebecca Brias, Amy Smith, Jill Temple, Duncan Jodrell, Alasdair Russell V Russell, Igor Chernukhin, Jason Carroll. Transcription factor switching drives progression of the classical subtype of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C071.