Project Summary Lung adenocarcinoma, the most common cause of cancer death worldwide, exhibits substantial heterogeneity in cellular identity, or differentiation state. In this disease, cancer cell identity correlates with critical clinical parameters, including patient prognosis, intrinsic sensitivity to therapy, and acquisition of drug resistance. Despite these correlations, major unanswered questions remain with respect to a) the regulatory networks controlling lung adenocarcinoma identity and (b) the mechanisms by which perturbation of these networks alters malignant potential. A major rationale for this proposal is that a mechanistic understanding of lineage specification in lung cancer must be achieved before differentiation state-specific therapeutic strategies can be developed. The immediate goal of this application is to test the central hypothesis that the transcription factors NKX2-1/TTF1, FOXA1 and FOXA2 coordinately regulate a gene network that controls lung adenocarcinoma differentiation and growth. This hypothesis will be tested in the following specific aims via an integrative experimental approach that employs genetically engineered mouse (GEM) models, analysis of primary human tumors, and functional studies in human cancer cell lines: (1) Characterize the role of FOXA1 and FOXA2 (FOXA1/2) in NKX2-1-positive lung adenocarcinoma. (2) Determine how global re-localization of FOXA1/2 after NKX2-1 loss impacts lung adenocarcinoma growth and differentiation. (3) Identify mechanisms by which NKX2- 1 represses non-pulmonary cell fates in lung adenocarcinoma.