PROJECT SUMMARY Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease that is incurable and progressive due to fibroblast activation, and the formation of scar tissue. Approximately 130,000 Americans suffer from IPF, with an estimated 50,000 new cases diagnosed each year. Although it is well accepted that myofibroblast accumulation is a central component of pathogenesis in IPF, the transcriptional program(s) that orchestrate fibroblast activation including fibroproliferation, fibroblast-to-myofibroblast transformation (FMT), survival, and collagen production are poorly defined and represent a significant knowledge gap in the field. WT1 is a zinc- finger transcriptional regulator, the function of which has been poorly studied in adult fibrotic lung diseases. We have recently reported direct clinical evidence of WT1 upregulation in fibroblasts of IPF and mouse models of severe fibrotic lung disease. Recently published studies from our lab highlight that WT1-positive fibroblasts play a pathogenic role in pulmonary fibrosis. In this regard, we generated fibroblast-specific WT1 knockout and overexpression mice to investigate mechanisms and develop new therapeutic interventions against WT1- driven pulmonary fibrosis. The focus of this application is to identify WT1-driven gene targets that are druggable to prevent fibroblast activation and pulmonary fibrosis. Our efforts to identify key targets of WT1 involved in fibroblast activation have led us to identify several anti-apoptotic genes, MYCN, and PLK1 as important mediators of WT1-induced pulmonary fibrosis. In support, we observed significant increases in MYCN and PLK1 by WT1 in lung fibroblasts of IPF and mouse models of pulmonary fibrosis. Importantly, we have identified a potent inhibitor of PLK1 called Volasertib (BI 6727; Phase I/II compound), as a lead small molecule inhibitor that can block a feed-forward loop of the MYCN-PLK1 axis to attenuate WT1-driven fibroblast activation. Together, these findings lead us to postulate that WT1 functions as a positive regulator of anti-apoptotic genes (BCL3 and BCL2L1), and the MYCN-PLK1 axis and that these factors are involved in fibroblast activation and pulmonary fibrosis. For this study, we propose three specific aims: 1) determine mechanisms by which WT1 inhibits apoptotic clearance in fibroblasts during the progressive expansion of fibrotic lesions; 2) determine mechanisms underlying WT1-driven the MYCN-PLK1 axis in fibroproliferation, FMT, and ECM production, and 3) test the therapeutic potential of volasertib therapy compared to FDA-approved anti-fibrotic therapies using two alternative mouse models of severe fibrotic lung disease. We will use advanced molecular methods and mouse transgenic approaches, coupled with detailed biochemical analysis of these WT1-driven processes in vivo and in vitro. Completion of the proposed experiments is likely to impart a significant understanding of WT1-driven fibroblast activation. The multidiscipl...