Defining the basal-like A-specific enhancer landscape in PDAC

Defining the specificity and P63-dependence of B-STEP

Supplementary Table S3 contains list of genes associated with Gö4Pdx4 super enhancers overlapping TP63.

Verification of P63-dependence and B-STEP enhancer-gene regulations.

Supplementary Table S4 contains L3.6pl TP63-dependent enhancer regions.

Supplementary Table S5 contains list of genes associated with L3.6pl TP63 dependent enhancers.

The file contains additional supplementary methods

Supplementary Table S2 contains GöPdx4 super enhancers regions overlapping TP63

Supplementary Table S6 contains list of qPCR primers used in the study.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and highly aggressive type of pancreatic neoplasm. Unfortunately, most patients will present with unresectable, locally advanced, or metastatic disease at the time of diagnosis. Current treatment methods (gemcitabine and nab-paclitaxel, FOLFIRINOX) are not sufficient for patients with late-stage disease and only modestly improve survival. Difficulties in treating PDAC lie in the characteristics of PDAC tumors. They are highly heterogeneous, have multiple signaling pathway alterations, and are highly metastatic with a dense tumor microenvironment (TME). Hence, PDAC remains one of the most lethal malignancies with a 5-year survival rate below 10%.The KRAS mutation is a hallmark of PDAC, occurring early in 95% of pancreatic neoplasms. The mutant KRAS protein constitutively activates the mitogen-activated protein kinase (MAPK) signaling pathway to sustain PDAC pathogenesis. Although the mutant KRAS gene is the initiating molecular alteration, chronic inflammation accelerates PDAC progression to a highly aggressive and therapy-resistant cancer. Our study shows that MAPK and the nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) inflammatory signaling pathway synergize to promote PDAC progression. Live cell imaging analysis of PDAC cell lines after the simultaneous hyperactivation of MAPK and NF-κB signaling by epidermal growth factor (EGF) and tumor necrosis factor alpha respectively (TNF alpha), showed significant increases in cell migration. An integrated genome-wide epigenetic and transcriptomic analysis (ChIPseq and RNAseq) after the simultaneous stimulation of both pathways revealed significant increases in the occupancy of the active mark, H3K27ac. This was observed around transcription start sites of a subset of genes involved in cell polarity, migration, and subtype switch, processes known to be important for tumor aggressiveness. Epigenome mapping of the transcription factors FOSL1 and RELA, which are downstream targets of the MAPK and NF-κB signaling pathways respectively, show their co-occupancy in the H3K27ac-enriched regions. Furthermore, loss-of-function approaches show the synergistic effect of both MAPK and NF-κB hyperactivation on the migration capacity in vitro and in vivo. Based on these observations, we aim to delineate the molecular mechanism involved in the synergism, investigate the role of the TME, and decipher the signaling interactions between PDAC tumor cells and cells of the TME. This study is the first comprehensive analysis of the synergism between MAPK and NF-κB signaling and will provide novel insights into the development of mechanism-based therapeutic approaches. Citation Format: Joana E. Aggrey-Fynn, Feda H. Hamdan, Alexander Q. Wixom, Thomas L. Ekstrom, Steven A. Johnsen. MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1481.

Objective Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. Design We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. Results iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. Conclusion Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.

AbstractA major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material.Implications:Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.