WT1 REGULATION OF PULMONARY FIBROSIS

项目摘要 特发性肺纤维化（IPF）是一种致命的纤维化肺病，由于肺纤维化， 成纤维细胞活化和瘢痕组织的形成。大约13万美国人患有IPF， 估计每年有5万个新病例被诊断出来。虽然肌成纤维细胞 蓄积是IPF发病机制的核心组成部分， 成纤维细胞活化，包括纤维增殖、成纤维细胞向肌成纤维细胞转化（FMT）、存活和 胶原蛋白的生产定义不明确，代表了该领域的重大知识空白。WT1是锌- 指转录调节因子，其功能在成人纤维化肺疾病中的研究很少。我们 最近报道了IPF成纤维细胞和小鼠模型中WT1上调的直接临床证据。 严重的纤维化肺病我们实验室最近发表的研究强调，WT1阳性成纤维细胞 在肺纤维化中起致病作用。在这方面，我们产生了成纤维细胞特异性WT1敲除， 过表达小鼠研究机制和开发新的治疗干预措施， 肺纤维化本申请的重点是鉴定WT1驱动的基因靶标， 可用于预防成纤维细胞活化和肺纤维化。我们努力确定WT1的主要目标 参与成纤维细胞活化的基因已经使我们鉴定出几种抗凋亡基因，MYCN和PLK1， WT1诱导的肺纤维化的重要介质。作为支持，我们观察到 MYCN和PLK1在IPF肺成纤维细胞和肺纤维化小鼠模型中的WT1。重要的是我们 已经确定了一种名为Volasertib（BI 6727; I/II期化合物）的PLK1强效抑制剂， 分子抑制剂，可以阻断MYCN-PLK1轴的前馈回路，以减弱WT1驱动的 成纤维细胞活化总之，这些发现使我们假设WT1的功能是积极的， 调节抗凋亡基因（BCL 3和BCL 2L1），和MYCN-PLK1轴，这些因素 与成纤维细胞活化和肺纤维化有关。在这项研究中，我们提出了三个具体的 目的：1）确定WT1抑制成纤维细胞凋亡清除的机制， 纤维化病变的进行性扩张; 2）确定WT 1驱动MYCN-PLK 1的潜在机制 纤维增生、FMT和ECM产生的轴，以及3）测试volasertib治疗的治疗潜力 与FDA批准的使用两种替代的严重纤维化肺小鼠模型的抗纤维化疗法相比， 疾病我们将使用先进的分子方法和小鼠转基因方法，再加上详细的 对这些WT 1驱动的过程进行体内和体外生化分析。完成建议 实验可能会赋予WT1驱动的成纤维细胞活化的重要理解。的 多学科团队将促进及时的方法，在肺部病理学的各个方面都有专业知识，并提供 深入了解IPF中急需的新治疗方法。

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 multidisciplinary team will facilitate a timely approach with expertise in all aspects of lung pathology and offers insight into new and highly needed treatments in IPF.