Lung cancer is the leading cause of cancer deaths worldwide. The most prevalent type of lung cancer is Non- Small Cell Lung Cancer (NSCLC). The goal of this application is to implement a collaborative effort involving preclinical models, bioengineering and functional genomics to further characterize and validate a novel “first in class” therapeutic strategy for the treatment of lung cancer. Our prior published work and extensive preliminary data indicates that blockade of CLCF1-CNTFR signaling represents a unique and previously unexplored approach for lung cancer therapy. The Sweet-Cordero and Cochran laboratories have collaborated extensively over the past several years to validate this signaling axis, first with shRNA genetic approaches, and now through the development of an engineered CNTFR receptor decoy (eCNTFR). In Aim 1, we will further develop eCNTFR as a therapeutic candidate by measuring its thermal and proteolytic stability, potential for immunogenicity and toxicity, manufacturability, and pharmacokinetics, and will optimize these properties as needed. We will also perform structural analysis of eCNTFR in complex with CLCF1 to define high affinity binding characteristics. Lastly, we will test eCNTFR for therapeutic efficacy across a wide array of preclinical models of NSCLC including human cell lines and patient-derived xenografts. In Aim 2, we will perform in vitro biochemical assays to fully define the cell-autonomous mechanism of action of eCNTFR blockade. To further understand the mechanism of action of eCNTFR in vivo, we will complement xenograft models with a well-characterized genetically engineered mouse model of lung cancer. Importantly, this model will allow us to study the effects of eCNTFR not only on tumor cells but also on the microenvironment, and to assess whether eCNTFR has immunomodulatory effects. In Aim 3, we will identify the most effective combination approaches to enhance eCNTFR therapy. These efforts will be carried out in a rational and unbiased manner by leveraging CRISPR/CAS9 using a library directed specifically at the targets of FDA approved drugs. Candidate combination therapies will then be tested in animal models. Our studies will elucidate critical biological underpinnings of this ligand/receptor signaling axis, and will provide preclinical validation of an innovative strategy for targeting lung cancer to warrant its further clinical development.