Project Summary: EGFR-mutant lung cancers (LCs) are initially highly responsive to EGFR inhibitors, but cancer adaptation resulting in drug resistance universally occurs. Acquired resistance mediated by lineage plasticity is particularly problematic; EGFR-mutant lung adenocarcinomas (ACs) can transform into either small cell (SC) or squamous cell (SQ) lung cancers. Understanding the molecular determinants of histologic transformation is critical to inform therapeutic strategies to block the emergence of new cell lineage states induced by cancer treatments. We have established that concurrent alterations in TP53 and RB1 are necessary but not sufficient to induce SC transformation in EGFR-mutant LCs; EGFR/TP53/RB1-mutant LCs have a 25% likelihood of transformation over time. In addition, we have assembled a cohort of resected mixed histology tumors (AC/SC and AC/SQ) that serve as a model of transformation where microdissection by histology isolates paired tumors representing pre- and post- transformation states. Using these complementary systems directly derived from patients, we will perform a mechanistic analysis of lineage plasticity utilizing EGFR/TP53/RB1-mutant LCs at high risk for transformation and mixed histology tumors that represent transformation in progress. Our central hypothesis is that while the somatic mutational landscape is critical in establishing conditions permissive of lineage plasticity, actual transformation to an alternative lineage is predominantly epigenetically driven and associated with consistent globally altered patterns of gene expression. Our first aim is to comprehensively molecularly characterize lineage plasticity using parallel whole exome, RNA and bisulfite sequencing focusing on patient samples from before, during (mixed AC/SC and AC/SQ) and after transformation. Resected mixed histology tumors (AC/SQ) will be microdissected and molecularly characterized as paired tumors. The second aim of the proposal is to investigate subclonal dynamics contributing to lineage plasticity using single cell RNA-sequencing. We will interrogate serial samples from our ongoing clinical trial to prevent transformation in patients with EGFR/TP53/RB1-mutant lung ACs and resected mixed histology AC/SC and AC/SQ tumors. Finally, we will utilize our patient-derived xenograft models of transformation to genetically and pharmacologically assess putative drivers of transformation, exploring rational interventional strategies. Our preliminary work has proposed initial targets (Wnt, EZH2, AKT, NOTCH) that will be expanded with findings from this proposal. Novel therapeutic interventions will be proposed based on our findings that can be rapidly translated to the clinical setting for LC and other disease states characterized by lineage plasticity.