B-233 Chromatin Accessibility Uncovers KRAS-driven FOSL2 Promoting Pancreatic Ductal Adenocarcinoma Progression Via Upregulating CCL28

Abstract

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal human cancer with 5-year survival rate less than 10%. Pancreatic cancer remains a malignancy of poor prognosis and effective therapies are lacking, therefore, studying the pathogenesis and identifying more therapeutic targets are urgently needed for PDAC treatment. The epigenetic mechanisms of PDAC progression are poorly understood. This study aims to identify key transcription factor (TF) by multiomics sequencing to explore the molecular mechanisms of TF with critical roles in PDAC. Methods we used ATAC-seq, H3K27ac ChIP-seq, and RNA-seq to characterized the epigenetic landscape by genetically engineered mouse models (GEMMs) of PDAC with or without KRAS and/or TP53 mutations. Survival of FOSL2 was assessed using Kaplan-Meier method and multivariate Cox regression analysis for patients with PDAC. We performed Cleavage Under Targets and Tagmentation (CUT&Tag) to study the potential targets of FOSL2. CCK8, transwell migration and invasion, RT-qPCR, western blot, IHC, ChIP-qPCR, dual-luciferase reporter, and xenograft model assays were used to explore the functions and underlying mechanisms of FOSL2 in PDAC progression. Results We found that the epigenetic changes were involved in some immuno-suppressed signaling with PDAC progression. Also, we identified a critical regulator FOSL2 which showed enriched open chromatin, H3K27ac signal, and higher RNA expression in KC and KPC than WT. The high expression of FOSL2 was associated with poor prognosis of patients with PDAC. Single-cell RNA-seq data showed that FOSL2 expression was very low in T, B, and acinar cells, and enriched in endothelial, ductal cells and upregulated in cancerous ductal cells compared with normal pancreatic ductal cells. functionally, FOSL2 can not only promote the proliferation, migration and invasion of pancreatic cancer, but also inhibit tumor immunity such as reducing the infiltration of CD8+T cells and increasing the infiltration of regulatory T (Treg) cells.In terms of mechanism, we found that CCL28 was a key target gene of FOSL2 by CUT&Tag sequencing. We confirmed that FOSL2 can regulate the expression of CCL28 by knockdown or overexpression of FOSL2 in vitro. ChIP-qPCR and dual luciferase reporter assays confirmed that FOSL2 activated the transcription of CCL28 by binding to the upstream of CCL28. In vivo mouse assay showed that FOSL2 promoted tumor growth and Treg cell recruitment through CCL28. We also explored the mechanism by which FOSL2 was upregulated in pancreatic cancer and found that KRAS mutation mediated FOSL2 transcription and subsequent CCL28 expression through the MAPK/ERK pathway, which implicated an immuno-suppressed regulatory axis of KRAS/MAPK-FOSL2-CCL28-Treg cells in PDAC development. Conclusion We described the changes of epigenetic landscape during PDAC progression and found that KRAS mutation promoted FOSL2 expression, which recruited more Treg cells by upregulating CCL28 and ultimately led to immunosuppression in pancreatic cancer. Targeting CCL28 with CCL28 antibody can reduce the invasion of Treg cells, which provides a new way to overcome the Treg cell-mediated tumor immune escape in patients with PDAC.