PROJECT SUMMARY Pulmonary fibrosis is a complex disease that limits lung function through development of collagen-rich scar tissue. Although there are currently two FDA-approved therapies for fibrosis patients, these drugs are capable of extending life only months and do not reverse the progression of the disease. The research described in this proposal would build upon evidence that reprogramming cellular fate through modulation of the epigenetic enzyme EZH2 can ameliorate several phenotypes of pulmonary fibrosis, including aberrant alveolar cell fate and pro-fibrotic cytokine signaling. Specifically, the aims will test if the FDA-approved EZH2 inhibitor, tazemetostat, will allow for prevention or reversion of these cellular phenotypes. The model systems to be used include both human and mouse organotypic cell cultures and two established in vivo models of pulmonary fibrosis, namely lung instillation of the chemotherapy bleomycin or adeno-associated virus (AAV) expressing TGFβ. For Aim 1, we will quantify the extent of aberrant basaloid/alveolar transition cells in lung epithelial cultures that are depleted for EZH2 activity genetically or pharmacologically, and assess rescue of this phenotype by deletion of the transcription factor FOXP2. FOXP2 deletion and knock-down will also be used to understand this important transcription factor’s role alveolar cell fate. We will also assess the down-regulation of IL1β by EZH2 inhibition, and its effects on both murine and human epithelial and fibroblast cells. The influence of TGFβ on fibroblasts and epithelial cells in the presence of EZH2 inhibitor will be tested. In Aim 2 in vivo lung injury models will be used to examine both prevention and treatment of lung fibrosis, and to characterize specific changes to the epithelial, fibroblast, and myeloid cell populations driven by EZH2 inhibition. Lung function testing, histology, scRNA-seq and flow cytometry will be used to characterize the lung phenotypes. Patient tissues will be used to understand if EZH2 activity is high and FOXP2 expression is low in aberrant basaloid/alveolar transition cells in vivo. We expect that this research could validate the use of EZH2 inhibition as a treatment for pulmonary fibrosis, and will expand our knowledge in the field of lung epigenetics and stem cell biology.