Project hypothesis and Specific Aims: We hypothesize that the molecular subtypes of EGFR and KRAS mutant NSCLC tumors with acquired mutation targeted drug resistance are associated with an immunosuppressive TME including both immune and non-immune stromal cells that can be therapeutically targeted in a way that will enhance the efficacy of drugs targeted to intrinsic resistance pathways. Specific Aim 1: Develop and characterize existing and new models of mutant EGFR and KRAS inhibitor acquired resistance including PDXs and PDX-derived organoids and characterize mechanistic relationships between molecular subtypes and the TME, including immune and non-immune stroma. Characterization will include histology, immunohistochemisry (IHC), RNAseq, and MS proteomics. We will identify specific molecular alterations that can be functionally tested and determine their effect on the TME. Examples being tested currently include 3-phosphoinositide-dependent kinase 1 (PDK1, PDPK1) expression, SETD1B inactivating mutations, and YAP (yes-associated protein) expression. We will test drugs that target specific cells in the TME to enhance the immune response. We are currently targeting M2 tumor associated macrophages (TAM). Osimertinib and G12C KRASi resistant PDXs and organoids will be interrogated to identify acquired drug resistance molecular pathways using MS proteomics, scRNAseq, and CRISPR screens. Specific Aim 2: Define treatment strategies in mutant EGFR and KRAS inhibitor acquired resistance models based on targeting pathways (e.g., PDK1, YAP, NF-B) implicated in current and future studies of acquired resistance caused by tumor-TME cell networks (e.g., CSFR1, CD74). Potential drug targets for overcoming acquired resistance to osimertinib (see preliminary data) include PDK1, YAP, NFkB, and TAM. We will develop therapeutic approaches based on TME characterization. H1975R has a more immune stimulatory TME than PC9 YAP thus providing a rationale for testing checkpoint blockade combined with PDK1 inhibition in H1975R. M2 polarized macrophages are increased in PC9 overexpressing wtYAP thus providing a rationale for testing TAM inhibition in PC9 YAP with a CSFR1 inhibitor. Residual tumors after osimertinib and KRAS G12Ci will be characterized following treatment using MS proteomics, scRNAseq, and CRISPR screens to identify novel acquired resistance pathways. The functional role of specific targets in acquired drug resistance will be validated using CRISPR KO. We will develop new models to include future novel drugs developed to target these and other pathways.