PROJECT SUMMARY Acquired resistance remains a significant obstacle to optimal therapeutic outcomes for patients with lung cancer. The long-term goals of project 3 are to elucidate mechanisms of acquired resistance to targeted therapies in lung cancer and to develop rational strategies to forestall/overcome resistance. During the past 5 years, we have successfully identified mechanisms of acquired resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) and developed therapeutic strategies to overcome resistance mediated by the EGFR T790M `second-site' mutation. We participated in the characterization of the mutant-selective third-generation EGFR TKI, osimertinib, which was recently approved by the US FDA for treatment of patients with metastatic T790M-positive EGFR-mutant lung cancer after progression on EGFR TKI therapy. Unfortunately, acquired resistance to osimertinib has already emerged in patients. Here, we plan to build on our extensive experience to characterize osimertinib resistance, in both the first-line setting (i.e., in the absence of T790M) and in the second-line setting (i.e., in the presence of T790M), to parallel ongoing clinical trials. In addition, using knowledge gained from our studies of EGFR TKI resistance, we will continue to advance our studies of acquired resistance to ALK TKI therapy in ALK-rearranged lung cancer. Therapeutic targeting of ALK fusion proteins with the first-generation ALK TKI, crizotinib, has shown significant clinical activity but is limited by the development of resistant disease. Although `next generation' ALK TKIs, including ceritinib, alectinib, ensartinib, and lorlatinib can overcome resistance to crizotinib, resistance to these more potent ALK TKIs has already developed in patients. We will leverage our proven proficiency in defining resistance mechanisms combined with innovative techniques – including forward genetic screens and siRNA screens – as well as unique resources – including novel cell lines and tumor biopsy samples taken at the time of disease progression on TKI therapy – to enhance our understanding of therapeutic resistance mechanisms with the overall goal of delaying or overcoming TKI resistance in lung cancer.