Epidermal Growth Factor Receptor (EGFR) mutant (mt) non-small cell lung cancer (NSCLC), initially has a high response rate to EGFR tyrosine kinase inhibitors (TKIs), however, resistance is inevitable. HGF-MET pathway activation and an epithelial-mesenchymal transition (EMT) transcription factor (TF) induced mesenchymal phenotype are commonly observed mechanisms of EGFR TKI resistance. We have identified the hepatocyte growth factor (HGF)-MET-TWIST1 axis as a novel targetable signaling axis that may account for both de novo and acquired resistance to EGFR TKIs including osimertinib. Our published data showed that the EMT-TF, TWIST1 is required for EGFR mt tumorigenesis and can mediated EGFR TKI resistance in vitro and in vivo through suppression of apoptosis. Targeting TWIST1, genetically or pharmacologically with our first-in-class TWIST1 inhibitor, harmine resensitized EGFR mt NSCLC to EGFR TKIs. Our preliminary data demonstrate that HGF increases TWIST1 expression and that TWIST inhibition can reverse HGF-MET mediated EGFR TKI resistance in vitro. Therefore, TWIST1 maybe the critical targetable node that connects these pathways. Central hypothesis: HGF-MET mediated induction of TWIST1 leads to the suppression of apoptosis and EGFR TKI resistance in EGFR mt NSCLC, which can be overcome with TWIST1 inhibition. We will test this hypothesis in the following Specific Aims: Aim 1: Determine the mechanism and clinical significance of HGF-dependent TWIST1 induction in EGFR mutant NSCLC. Hypothesis: HGF leads to increased TWIST1 expression and activity through phosphorylation by ERK and/or AKT (Aim 1a). We further hypothesize that HGF-MET activation correlates with increased TWIST1 expression and poor response to EGFR TKIs in the EGFR TKI de novo and acquired resistance setting in patients (Aim 1b) Aim 2: Elucidate the mechanism of HGF-TWIST1-mediated EGFR TKI resistance. Hypothesis: TWIST1 mediates HGF-MET dependent EGFR TKI resistance in both de novo and acquired resistance through suppression of apoptosis in vitro (aim 2a) and in vivo (aim 2b-c). Aim 3: Evaluate the efficacy of the TWIST1 inhibitor, harmine to overcome HGF-MET induced EGFR TKI resistance. Hypothesis: Targeting TWIST1 with harmine in combination with osimertinib will overcome HGF- induced resistance as well as MET driven resistance in the acquired resistance setting in vitro and in vivo. Translational Impact: There are no FDA approved targeted therapies after progression on osimertinib. We are uniquely positioned to identify TWIST1 as a mediator of HGF-MET-dependent EGFR TKI resistance and a novel therapeutic target for the treatment of EGFR TKI resistance.