Project Summary Osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), is the current standard of care for first-line therapy for EGFR mutant lung cancer patients. While osimertinib is effective in inducing tumor regressions, improving quality of life and prolonging survival in most patients, it is not curative. Early studies of acquired resistance suggest that even after standard histologic and genomic assessments for resistance mechanisms, a substantial portion of osimertinib resistance remains uncharacterized. This incomplete understanding of osimertinib resistance poses a significant barrier to developing new and effective therapies for clinical use. We hypothesize that, in contrast to earlier generation EGFR inhibitors, non-genomic mechanisms are driving the majority of acquired resistance to first-line osimertinib. To date, assessments of EGFR TKI acquired resistance mechanisms have focused on genomic resistance mutations, amplifications, and fusions, in part because these can be readily detected in tumor tissue or in blood-based ctDNA liquid biopsies. In this project we seek to discover the spectrum of non-genomic osimertinib resistance. Our main objective is to identify both tumor intrinsic mechanisms of osimertinib resistance as well as tumor extrinsic microenvironmental resistance mechanisms. In Aim 1, we will use RARE-Seq, a novel blood-based assay to quantify tumor gene expressions patterns of resistance in EGFR mutant NSCLC patients relapsing on osimertinib. In Aim 2, we will investigate tumor cell extrinsic mechanisms of osimertinib resistance by leveraging our robust translational infrastructure to develop cancer-associated fibroblast models from osimertinib resistance biopsies. We will use these models to dissect functional interactions between CAFs, tumor cells and immune cells, and we will integrate these results with spatial molecular analysis of clinical biopsies. Collectively, the studies described in this proposal will generate a comprehensive picture of osimertinib resistance and set the stage for future development of therapies that overcome resistance to osimertinib.