Unresectable locally advanced non-small cell lung cancer treated with concurrent platinum-based chemotherapy and thoracic radiation results in primary tumor recurrence in >35-80% of cases from radioresistant tumors. Therefore, research aimed at understanding mechanisms of radioresistance and strategies to overcome this problem are paramount to better lung cancer treatment outcomes. We found TWIST1 was commonly overexpressed in human lung cancers and Twist1 could confer radiation resistance to lung epithelial cells, lung cancer cells and autochthonous mouse lung tumors in vivo. Twist1 is a basic helix- loop-helix transcriptional factor. The domains of Twist1 and transcriptional targets of Twist1 that are required for radioresistance are unknown. In order to maximize the therapeutic potential of inhibiting Twist1 for lung cancer, we must precisely define the protein domains of Twist1 and/or the downstream transcriptional targets of Twist1 required for radioresistance. In the present proposal we have three specific aims to test two hypotheses that will provide more mechanistic insight into the structure-function relationships of Twist1 and lung cancer radioresistance. One central hypothesis of this proposal is that specific protein domains of Twist1 are required for radioresistance. We have developed a novel lung epithelium specific Twist1 mouse model and various mutant versions of Twist1 to facilitate these studies. Our second testable hypothesis is there is a defined set of core transcriptional targets of Twist1 required for radioresistance. The studies outlined in our proposal will provide valuable knowledge to the pathophysiologic role of Twist1 in lung cancer and Twist1- induced radioresistance. Finally, validating a novel preclinical inducible lung cancer model of radioresistance will create a model to test new treatments for this fatal disease in combination with radiation. Specific Aim #1: Characterize the domains of Twist1 required for radioresistance in vitro. We will use mouse embryo fibroblasts (MEFs) and isogenic human lung cancer cells expressing Twist1 and Twist1 mutants to examine which domains of Twist1 are necessary for a radioresistant phenotype. We will determine the cell autonomous mechanisms of Twist1-induced radioresistance in vitro. Specific Aim #2: Characterize the domains of Twist1 required for radioresistance of KrasG12D-induced autochthonous lung tumors. We will test this hypothesis with serial non-invasive imaging and confirm our findings with standard molecular-histologic methods to follow the response to radiation in our novel Twist1 transgenic lung tumor model. We will also examine the dominant mechanisms for Twist1-dependent radiation response modulation in vivo. Specific Aim #3: Determine Twist1 transcriptional targets correlated with Twist1-induced radioresistance using gene expression and ChIP-chip profiling. We will use integrative genome-wide bioinformatic approaches on MEFs and human lung cancer cells to isolate the core gene targets required for Twist1-induced radioresistance.