WT1 REGULATION OF PULMONARY FIBROSIS

项目总结 特发性肺纤维化(IPF)是一种致命的纤维性肺部疾病，由于 成纤维细胞活化，并形成瘢痕组织。大约有13万美国人患有IPF，其中 据估计，每年新诊断的病例有5万例。尽管人们普遍认为肌成纤维细胞 积聚是特发性肺间质纤维化发病机制的中心组成部分，它是转录程序(S)协调的 成纤维细胞的激活包括成纤维细胞增殖、成纤维细胞到肌成纤维细胞的转化(FMT)、存活和 胶原蛋白的生产定义不明确，在该领域存在着重大的知识差距。WT1是一种锌- Finger转录调控因子在成人肺纤维化疾病中的作用研究较少。我们 最近报道了IPF成纤维细胞和小鼠模型WT1上调的直接临床证据 严重的纤维性肺病。我们实验室最近发表的研究强调了WT1阳性的成纤维细胞 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的WT1基因敲除和 高表达小鼠研究WT1-机制并开发新的治疗干预措施 导致肺纤维化。这项应用的重点是识别WT1驱动的基因靶点 可预防成纤维细胞活化和肺纤维化的药物。我们为确定WT1的主要目标所做的努力 参与成纤维细胞的激活，使我们发现了几个抗凋亡基因，MYCN和PLK1 WT1诱导肺纤维化的重要介质。在支持方面，我们观察到 肺纤维化模型小鼠肺成纤维细胞中WT1对MYCN和PLK1表达的影响重要的是，我们 已经确定了一种名为Volasertib的PLK1有效抑制剂(BI 6727；I/II期化合物)，是一种小剂量的铅 一种分子抑制剂，可以阻断MYCN-PLK1轴的前馈环，以减弱WT1驱动的 成纤维细胞激活。综上所述，这些发现使我们假设WT1发挥着积极的作用 抗凋亡基因的调节因子(BCL3和BCL2L1)和MYCN-PLK1轴以及这些因子 参与成纤维细胞活化和肺纤维化。对于这项研究，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 纤维化病变的渐进性扩张；2)确定WT1驱动的MYCN-PLK1的机制 Axis在纤维增殖、FMT和ECM产生中的作用，以及3)测试Volasertib疗法的治疗潜力 与FDA批准的使用两种可选的严重纤维化小鼠模型的抗纤维化治疗进行比较 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 在体内和体外对这些WT1驱动的过程进行生化分析。完成拟议的 实验可能会使人们对WT1驱动的成纤维细胞激活有重要的了解。这个 多学科团队将利用肺部病理的所有方面的专业知识促进及时的方法，并提供 洞察IPF中迫切需要的新治疗方法。

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.