PROJECT ABSTRACT Cell identity, crucially controlled by transcription factors (TFs), is perturbed in cancer, contributing to highly aggressive and metastatic tumors, such as pancreatic ductal adenocarcinoma (PDAC). PDAC represents a formidable challenge with an alarmingly low 5-year survival rate of less than 10%. Predominantly driven by mutations in KRAS and subsequent loss of tumor suppressor genes, PDAC is poised to become the second leading cause of cancer-related deaths by 2030. Integrated genomic, transcriptomic, and proteomic analyses have identified two distinct PDAC subtypes: classical and basal. The critical difference between these subtypes lies in the expression of endodermal lineage specifiers, with the classical subtype expressing FOXA1, FOXA2 (FOXA1/2), and HNF4⍺, critical for pancreatic cell-fate determination, while the basal subtype downregulates these genes, leading to the loss of endodermal identity. The clinical significance of these subtypes is evident as the basal subtype confers shorter median survival and exhibits differential responses to first-line chemotherapies. We reason that endodermal lineage-specific TFs not only serve as a biomarker for the classical subtype but are essential regulators of PDAC subtype differentiation. Understanding the molecular regulators of the classical subtype is crucial as they may hold the key to developing effective targeted therapies and improving patient outcomes. Furthermore, the emergence of targeted therapies has highlighted the role of changes in cancer cell identity as a resistance mechanism. Here we focus on the pivotal transcription factors, FOXA1/2 and HNF4α, that play critical roles in pancreatic development. Despite their relevance in other cancers, their specific functions in PDAC remain largely unexplored. In this study, we aim to define the functional roles of FOXA1/2 and HNF4⍺ in the classical PDAC subtype, exploring their regulation of growth and cellular identity. Additionally, we will investigate how MEK/ERK signaling influences FOXA1/2 and HNF4⍺'s transcriptional activity in PDAC, shedding light on the mechanisms underlying lineage switching upon MEK/ERK inhibition. Overall, we will test the central hypothesis that FOXA1/2 and HNF4⍺ are critical regulators of the transcriptional network governing the cellular identity of classical PDAC and that MEK/ERK signaling modulates the activity of these TFs. To test this hypothesis, we will be reconstituting and modulating the expression of these factors in human and murine PDAC cell and organoid lines and assessing gene expression changes and genomic localization of these TFs through ChIP-seq. This proposal will show how oncogenic signaling and lineage- defining TFs regulate cancer cell identity.