Molecular subtyping of pancreatic ductal adenocarcinoma (PDAC) has defined two major transcriptional subtypes, Classical and Basal-like, which are presently the most powerful predictors of patient outcomes. Thus, there is a critical need to determine the underlying mechanisms responsible for PDAC cell identity. Our preliminary experiments demonstrate that inactivation of PHF2, a histone demethylase, in low-passage patient-derived Classical PDAC cells results in a striking loss of the Classical gene program and virtually abolishes tumor growth. In addition, deletion of Phf2 suppresses pancreatic tumor initiation and prolongs survival in genetically engineered mouse models. Moreover, our CUT&RUN experiments reveal that PHF2 co-localizes on chromosomes with CTCF and SMC3, proteins that contribute to cell identity by regulating the three-dimensional architecture of chromatin. Based on these exciting preliminary results, our central hypothesis is that PHF2, controls the Classical gene program in PDAC by regulating three-dimensional architecture of chromatin. Experiments in this proposal will employ a unique collection of low-passage human cell lines, high resolution mapping of chromatin-associated proteins and chromatin architecture, and NanoString GeoMx spatial transcriptomic analysis of genetically engineered mouse models to define the role of PHF2 in PDAC. Three specific aims are proposed to test the central hypothesis: 1) Define the subsets of PDAC that are dependent on PHF2; 2) Identify the mechanisms by which PHF2 regulates gene expression in PDAC; and 3) Define the contribution of Phf2 to tumorigenesis in genetically engineered mice. In the first aim, orthotopic xenograft tumor models allowing for the temporal inactivation of PHF2 by CRISPR/Cas9 will be employed to determine the importance of PHF2 in the maintenance of PDAC tumors as well as their response to chemotherapy. In the second aim, CUT&RUN, HiChIP, and RNA-seq experiments will be employed to define the global localization of PHF2 on chromatin in PDAC cells and ascertain its role in recruiting CTCF and cohesion to DNA to mediate DNA loop formation. For the third aim, genetically engineered mouse models of PDAC lacking Phf2 will be used to elucidate its role in the KRAS-driven changes in cell identity required for pancreatic acinar cells to transform to invasive cancer . Spatial transcriptomic studies will be performed to ascertain how Phf2 deficiency alters the composition of the tumor microenvironment by the development of PDAC that is deficient in the Classical program. The research proposed in this application is innovative because it will define a novel function for PHF2 in regulating chromatin architecture, further elucidating the mechanisms that allow for cell type-specific chromatin architecture. The proposed research is significant because it will provide strong evidence for the role of epigenetic regulation of PDAC cell identity, ultimately providing new opportunities for the ...