Abstract B034: Characterizing the role of HNF4α P1 and P2 isoforms in the classical subtype of pancreatic ductal adenocarcinoma

Abstract

Abstract Background: Recent transcriptomic studies have identified distinct subtypes of pancreatic ductal adenocarcinoma (PDAC). Broadly, PDAC can be classified into a classical or basal-like subtype. The classical subtype has a better response to chemotherapy and expresses higher levels of endodermal lineage specifiers compared to the basal-like subtype. However, the field lacks a comprehensive understanding of the key subtype regulators. Functional analysis of transcription factors associated with each subtype is needed to elucidate their roles in the regulation of PDAC subtypes and to identify therapeutically targetable vulnerabilities. Previous work in our lab demonstrated that HNF4α is a key regulator of the classical subtype. Using genetically engineered mouse models, our lab showed that loss of HNF4α leads to an increase in tumor burden, a shift from glandular to poorly differentiated histology, and a decrease in the classical subtype score. However, while murine PDAC only expresses one set of HNF4α isoforms, human PDAC expresses both sets of HNF4α isoforms, suggesting murine PDAC may not fully recapitulate human disease. There are 2 subsets of HNF4α isoforms driven by expression from 2 distinct promoters, P1 and P2. P1 and P2 isoforms are differentially expressed within the gastrointestinal (GI) tract and play dichotomous roles in GI malignancies. In hepatocellular carcinoma and colorectal cancer, expression of P1 isoforms significantly restrained tumor growth while expression of P2 isoforms was permissive for tumor growth. In HNF4α-positive human PDAC, P2 isoforms are always expressed but there is variable expression of P1 isoforms. Based on the dichotomous role of HNF4α isoforms in both normal tissue and cancer, we hypothesize that in HNF4α-positive PDAC, P1 isoforms will restrain tumor growth and promote a distinct differentiation state compared to P2 isoforms. Methods: To investigate HNF4α isoforms in human PDAC, we exogenously expressed P1 and P2 isoforms in HNF4α-negative PDAC cell lines. To complement our exogenous studies, we developed CRISPRi methods to selectively knockdown expression of endogenous P1 or P2 isoforms at each promoter. We then performed functional assays to measure growth and RNAseq to characterize transcriptional changes. Results: Immunohistochemical analysis of human samples confirmed that HNF4α-positive tumors uniformly express P2 but variably express P1 isoforms. Preliminarily, we found that exogenous P1 significantly restrained growth in two PDAC cell lines compared to exogenous P2. RNAseq in a PDAC cell line showed that P1 is a stronger transcriptional activator than P2, consistent with published data. GSEA found that P1 induced a stronger intestinal signature compared to P2. Future Directions: Initial data demonstrated dichotomous functional roles for P1 and P2 isoforms in human PDAC further supporting the need to investigate HNF4α at the isoform level. Subsequently, we will investigate the functional role of endogenous HNF4α via a CRISPR competition assay, RNAseq, and ChIPseq in 2D cell lines and organoids. Citation Format: Pengshu Fang, Walter A. Orellana. Characterizing the role of HNF4α P1 and P2 isoforms in the classical subtype of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B034.