PROJECT ABSTRACT. Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) the predominant histologic subtype of lung cancer and lung adenocarcinoma the major subset of NSCLC. Despite clinical progress with the use of targeted therapies, drug resistance remains a problem that limits patient survival. A less well understood aspect of the evolution of drug resistance is the drug tolerant, “persister” cell state, in which a subpopulation of cancer cells survives initial targeted treatment to form a minimal residual disease (MRD) reservoir that is a precursor to acquired resistance. We propose an innovative, multidisciplinary and collaborative project to hopefully improve the survival of NSCLC patients by defining the role that the Hippo-YAP pathway plays in promoting drug tolerance and MRD to current pathway targeted therapies. Extending work that we completed in the first funding cycle of this R01, we aim to capitalize on our discovery of the Hippo-YAP signaling pathway as a critical molecular circuit and therapeutic target in the many cancers driven by hyperactivation of RTK-RAS-MAPK signaling. Our data suggest an emerging paradigm in which YAP activation is a key functional feature of the drug tolerant state during RTK-RAS-MAPK targeted inhibition in NSCLC. We observed YAP activation during therapy in multiple oncogene-driven NSCLC preclinical models treated with EGFR, ALK, KRAS and SHP2 inhibitors. YAP upregulation promoted the expression of several survival factors including BCL-xL and RTKs such as FGFR1 and ErbB2. Furthermore, through single- cell RNA sequencing (scRNAseq) performed longitudinally in clinical specimens obtained from patients treated with targeted inhibitors, we discovered that residual cancer cells often show lineage plasticity and a transition to an alveolar type (AT)1/2-like transcriptional state that is a novel phenotype of slow cycling, drug tolerant cancer cells. We verified this occurred in bona fide cancer cells (i.e., not from misannotated normal alveolar cells) and was distinct from therapy-naïve adenocarcinoma or normal AT1/2 cells. Our data suggest a role for YAP in promoting drug tolerance and this novel form of lineage plasticity. Inhibition of YAP signaling with YAP/TEAD small molecule inhibitors that are in clinical development suppressed drug tolerant cancer cell survival and expression of molecular markers of the AT1/2-like lineage switch. We propose to further test the hypothesis that YAP signaling is a key molecular switch that regulates the biological and clinical response to RTK-MAPK pathway inhibitors. In Aim 1, we define the role of YAP in promoting drug tolerance and the AT1/2-like lineage plasticity present in drug tolerant cancer cells in RTK-RAS-MAPK-driven NSCLCs. We also test whether pharmacologic inhibition of YAP/TEAD can thwart MRD and enhance response to RTK-RAS inhibitors in preclinical models. In Aim 2, we study molecular features of YAP/TEAD act...