ABSTRACT Lung cancer is the leading cause of cancer-related deaths in the U.S., with ~236,700 new cases and ~130,200 deaths estimated for 2022. A joint effort by both PIs in this grant led to the identification of the oncogenic kinase protein kinase C epsilon (PKCe) as a key player in lung carcinogenesis. PKCe is aberrantly up-regulated in lung adenocarcinoma and is associated with poor outcome in patients specifically harboring KRAS mutations. Using genetically engineered mouse (GEM) models, we demonstrated that PKCe is required for both carcinogen- and Kras-driven lung tumorigenesis. In addition to this role in cancer initiation, our work established novel roles for PKCe in cellular events associated with late cancer stages. Indeed PKCe is a major player in lung cancer cell motility and invasion via activation of the small G-protein Rac1, linking this kinase to metastatic dissemination. That PKCe plays critical permissive roles first during adenoma initiation and then again later during the acquisition of metastatic competency highlights the functional complexities of the PKCe signaling events in lung cancer. Interestingly, CRISPR-mediated inactivation of PKCe in the initiating cell-of-origin does not significantly affect Kras- G12D-induced tumor development, leading us to hypothesize that PKCe does not strictly act in a tumor cell autonomous manner to permit oncogenic KRAS-mediated tumorigenesis. To test this hypothesis, in Aim 1 we will generate and characterize a series of GEM models to restrict genetic deletion of PKCe to either oncogenic Kras- expressing cancer cells or to diverse stromal cell types present in the tumor microenvironment, including non- cancerous epithelial, mesenchyme and hematopoietic cells. Gene expression studies on isolated cells using fluorescence-based lineage tracing techniques will provide significant mechanistic insights. In Aim 2, we will thoroughly dissect the mechanistic basis of motility/invasive signaling activation by PKCe. Our hypothesis is that PKCe activates Rac1-mediated formation of cell ruffles and motility in KRAS mutant lung cancer cells via Rac Guanine nucleotide Exchange Factors (Rac-GEFs). We will identify and characterize candidate Rac-GEFs as PKCe effectors responsible for this phenotype. In Aim 3, we will establish the involvement of PKCe and its effector Rac-GEFs for the development of metastatic lung cancer. We will use combined in vitro and in vivo approaches to pin down mechanistic defects in the metastatic cascade upon CRISPR-mediated deletion of PKCe and Rac-GEFs in lung adenocarcinoma cells. We will establish GEM models and use lentiviral CRISPR-based approaches to determine the permissive contribution of PKCe and its GEF effectors to lung adenocarcinoma metastasis, and identify relevant gene expression and signaling signatures contributing to this phenotype. Thus, by using innovative, state-of-the-art genetic and molecular approaches, our studies should reveal the vast multidimensional...