PROJECT ABSTRACT: Lung adenocarcinoma (LUAD) is the largest subtype of lung cancer and is the most common cause of cancer related death. LUAD progression is driven by a combination of genetic alterations and epigenetic changes that confer increased plasticity and heterogeneity, but underlying mechanisms dictating cell fate and plasticity in LUAD remain poorly understood. Interestingly, the lineage defining transcription factors, NKX2-1 and FoxA1/2, coordinately maintain a pulmonary identity in LUAD. In the absence of NKX2-1, FoxA1/2 can activate gastric transcription factors including HNF4α to substantially alter the differentiation state of LUAD. Recent work suggests that NKX2-1-positive (NKX2-1+) LUAD evolves from an alveolar-like state and adopts various disparate cell fates that drive progression, including “mixed lineage” states, characterized by acquisition of gastrointestinal (GI) and pulmonary-like states in the same cell. This proposal will investigate how these gastric states are maintained and how they may promote LUAD tumorigenesis. HNF4α levels increase over the course of LUAD progression, and HNF4α and NKX2-1 are co-expressed in a subset of human and mouse NKX2-1+ LUAD tumors. This supports a putative model by which HNF4α drives LUAD progression and promotes a mixed-lineage state by competing with NKX2-1 for FoxA1/2 binding. Additionally, given previous findings that MAPK signaling is important for LUAD progression and alveolar fate, and that NKX2-1 is known to be phosphorylated and inactivated by ERK, MEK/ERK signaling may promote mixed-lineage states by destabilizing NKX2-1/FoxA1/2 interactions and favoring HNF4α/FoxA1/2 binding. I hypothesize that HNF4α promotes LUAD tumorigenesis and proliferation, and that MAPK signaling modulates the mixed-lineage state by altering the activity and interactions of key lineage specifiers. To test this hypothesis, I will modulate MAPK signaling using complementary pharmacological and genetic approaches and assess expression of key mixed lineage genes, as well as visualizing protein complexes upon MAPK manipulation using a Proximity Ligation Assay. Additionally, I will perform ChIP-seq to determine how modulating MEK/ERK alters NKX2-1 and FoxA1/2 chromatin binding at gastric and pulmonary targets. I will also expand on preliminary data from our group demonstrating that HNF4α drives tumorigenesis and proliferation in murine organoids as well as a KRAS-driven LUAD mouse model. I will genetically modulate HNF4A in human LUAD cell lines expressing both NKX2-1 and HNF4A, perform proliferation assays, and create cell line xenograft models to measure tumor growth. I will perform integrative RNA-seq and ChIP-seq analysis to understand key factors underlying HNF4α-mediated growth and progression in NKX2-1+ LUAD. Completion of these aims will unveil key insights into the mechanisms by which LUAD heterogeneity drives tumor growth and progression, knowledge which will be critical for development of effective...