Project Summary/Abstract: The goal of this translational Project within BAATAAR-UP is to characterize the mechanisms of, and therapeutically counteract, acquired resistance to molecular therapies in non-small cell lung cancer (NSCLC) by delineating the tumor-tumor microenvironment (TME) ecosystem and its plasticity during treatment. Acquired resistance (AR) is defined as tumor progression that occurs during active therapy and after an initial therapy response. The overarching hypothesis is that AR can be therapeutically counteracted by defining the cellular and signaling networks allowing tumors to survive and grow during therapy. The use of molecularly targeted therapies that inhibit oncogenic driver alterations such as mutant EGFR and KRAS and block immunosuppressive checkpoints such as PD1/L1 is improving outcomes for patients with aggressive tumors including NSCLC, which nonetheless remains the leading cause of cancer mortality. Despite profound progress, a major challenge to transforming NSCLC into a chronic or curable cancer is AR that enables lethal tumor progression in patients. Understanding the mechanisms driving AR is essential to develop strategies to counteract it and induce sustained anti-tumor responses to improve patient survival. Critical knowledge gaps are whether and how tumor cell/TME cell interactions and spatial relationships promote AR. Another aspect of the evolution of AR that is poorly defined is the basis of the incomplete response and residual disease that is typical during therapy. This residual disease contains drug tolerant cancer cells and interactive TME cells that evolve together to promote the aggressive transition into AR. Defining how this transition occurs could provide strategies to thwart it. Our work accomplished during the prior U54 funding period showed that oncogene-driven NSCLCs contain a rich cellular ecosystem that evolves during molecular treatments (e.g., EGFR, ALK, and RAS pathway targeted agents). We discovered heterogeneity and plasticity in tumor cells and TME cells, including immune and non-immune cell types, and spatial relationships at different clinical treatment states including at AR that we hypothesize contribute collectively to AR. These include bi-directional interactions between tumor macrophages and cancer cells and tumor fibroblasts and cancer cells via discrete signaling circuits that promote cancer cell survival and remodel the TME into a more pro-tumor phenotype at AR. Examples include cytokine (CSF1, TNFa, IL1b), and CD47 signaling between tumor macrophages and cancer cells and macrophage migration inhibitory factor (MIF)-CD74/CD44 and extracellular matrix (ECM)/integrin signaling between tumor fibroblasts and cancer cells at AR. Our goal is to define and therapeutically target these, and additional, cancer cell and TME cell networks to therapeutically thwart AR. We focus on clinically important and prevalent NSCLC subtypes defined by oncogenic mutant EGFR and KRAS and current clinical targeted inhibitors against these major oncogenic drivers. Two Specific Aims are proposed. Our work will highlight mechanisms driving AR across the tumor-TME continuum in EGFR- and KRAS-driven NSCLCs and identify counteracting therapeutic strategies for translation.