PROJECT SUMMARY The overall goal of Project 3 of this U54 application is the organoid-based definition of mechanisms of acquired resistance (AR) to targeted therapy in non-small cell lung cancer (NSCLC). The treatment of NSCLC employs numerous therapies directed against the EGFR, ALK, KRAS G12C, ROS1 and PD-1/PD-L1 pathways. However, despite initial responses, these molecularly-targeted therapies are not curative, and virtually all patients eventually succumb to progressive disease. Acquired resistance features prominently in relapse, such as with EGFR T790M or C797S mutations following gefitinib or osimertinib treatment; however, known AR mechanisms cannot be identified for a significant proportion of patients receiving targeted therapy. Further compounding this dilemma are the distinct contributions of tumor-intrinsic versus tumor microenvironmental (TME) influences. The TME in particular contains extracellular matrix (ECM), fibroblast, vascular and immune components that can vitally impact the development and manifestations of AR. The study of TME-dependent and -independent mechanisms of AR have been further hindered by a distinct lack of in vitro human experimental systems allowing holistic recapitulation of the TME in patient biopsies. Our application leverages the synergistic strengths of the Sourav Bandyopadhyay and Calvin Kuo groups in organoid and proteomics technology to address mechanisms of EGFR and KRAS G12C AR in NSCLC. We conduct advanced proteomic approaches and organoid culture both from primary tumor biopsies at acquired resistance (from Project 1) as well as from patient-derived xenograft (PDX) tumors grown in human immune reconstituted mice (from Project 2). Our organoids are either “epithelial-only” or novel air-liquid interface (ALI) organoid cultures that co-preserve tumor epithelium en bloc with tumor-infiltrating fibroblasts and immune subsets (T, B, NK, myeloid). Aim 1 leverages the results of a NSCLC organoid CRISPR screen to determine if osimertinib-sensitizing loci can overcome, delay or alter acquired resistance in clinical biopsy/PDX AR organoids, emphasizing hits with TME mechanisms. Aim 2 uses our mass spectrometry platforms to characterize total proteome and phospho-proteomic changes in treatment-naive, residual and acquired resistance organoids and PDX, while pursuing lead candidate TROP2 and new target nominations by genetic, mechanistic and pharmacologic studies. Lastly, Project 1 has identified MIF/CD74/CD44 and CD47/SIRPa tumor-fibroblast- macrophage pathways upregulated during AR which are then functionally explored in Aim 3 using ALI organoids from AR clinical biopsies and PDX tumors. Overall, we propose a comprehensive, translational approach that exploits complementary organoid and proteomics expertise to directly study biopsies and PDX from NSCLC patients with acquired resistance to targeted therapy, towards development of improved therapeutic approaches.