Translational Dysregulation Driving Mesenchymal Cell Fibrogenic Transformation and Chronic Lung Allograft Dysfunction

项目摘要/摘要 以进行性移植失败为特征的慢性肺移植功能障碍(CLAD)发生在50%的肺移植患者中 这是这种救命手术后长期效果不佳的主要原因。 主要临床表现为持续性阻塞性呼吸缺陷，称为毛细支气管炎。 闭塞症(BOS)由小气道纤维性闭塞或闭塞性细支气管炎(BO)引起。 移植物驻留间充质细胞在BO发病机制中起关键作用及我们最近的研究 证明MC的自主激活以增加基质沉积和稳定激活为标志 促纤维化途径是同种异体移植物纤维化和功能障碍的关键驱动因素。不同的信号通路是 显示在真核细胞翻译起始因子4E(EIF4E)水平上聚集以调节关键的纤维化 其功能类似于BOS MC中I型胶原的表达。在这项应用中，我们旨在研究MAP激酶的作用- 相互作用的丝氨酸/苏氨酸激酶(MNK)诱导维持过程中丝氨酸209位eIF4E的直接磷酸化 BOS单核细胞的纤维化转化及MNK/eIF4E通路在发病机制中的作用 移植后BO的发生率。MNK激酶诱导的丝氨酸209位eIF4E的磷酸化已被证实 在促进其致癌潜力方面具有关键作用，但尚未在纤维化背景下进行研究。我们的 初步数据显示，患者MC中有较高的丝氨酸209位eIF4E磷酸化 有了BOS。EIF4E磷酸化S209被发现在调节关键的纤维形成蛋白自体趋化蛋白和 维持BOS单核细胞的纤维化功能。MNK信号在BOS细胞中上调，遗传性或 药物靶向MNK活性抑制eIF4E磷酸化S209和BOS-MCs的纤维化功能 体外培养。体内应用MNK抑制剂EFT-508可减轻小鼠原位移植肝纤维化 肺移植模型。在目标1中，我们将利用患者±BOS来源的MC来剖析分子 EIF4E Ser209磷酸化促进人肺纤维化转化的机制 MCS。目的2研究MNK/eIF4E信号通路在小鼠原位肺BO发生发展中的作用 利用MNK1/2基因敲除和eIF4ES209A/A突变小鼠靶向MNK/eIF4E S209建立BO移植模型 在供体肺中。新型口服MNK1/2抑制剂eFT508在小鼠原位全肝的临床前研究 Aim 3中提出的评估纤维化和免疫/炎症终点的肺移植BO模型 将为未来的人类治疗试验奠定基础。总而言之，这些研究将为 MNK/eIF4E Ser209磷酸化在肾小球系膜细胞纤维化活化中的作用及其潜在的诱导性 目的是阻止包膜的纤维化进展和发展。

PROJECT SUMMARY/ABSTRACT Chronic lung allograft dysfunction (CLAD) marked by progressive graft failure develops in 50% of lung transplant recipients by 5 years and is the major cause of poor-long term outcomes after this life-saving procedure. Predominant clinical presentation is development of persistent obstructive ventilatory defect termed bronchiolitis obliterans syndrome (BOS) resulting from fibrotic obliteration of the small airways or bronchiolitis obliterans (BO). Graft-resident mesenchymal cells (MCs) play a key role in pathogenesis of BO and our recent studies demonstrate that autonomous activation of MCs marked by increased matrix deposition and stable activation of pro-fibrotic pathways is a key driver of the allograft fibrosis and dysfunction. Various signaling pathways were shown to converge at the level of eukaryotic translation initiation factor 4E (eIF4E) to regulate key fibrotic functions like collagen I expression in BOS MCs. In this application we aim to investigate the role of MAP kinase– interacting serine/threonine kinase (MNK) induced direct phosphorylation of eIF4E at serine 209 in maintaining fibrogenic transformation of BOS MCs and determine the contribution of MNK/eIF4E pathway to pathogenesis of BO post-transplantation. MNK kinase induced phosphorylation of eIF4E at serine 209 has been demonstrated to be critical in promoting its oncogenic potential but has not been investigated in the context of fibrosis. Our preliminary data demonstrates a constitutively higher eIF4E phosphorylation at Serine209 in MCs from patients with BOS. eIF4E phospho-S209 was found to be critical in regulating key fibrogenic protein autotaxin and sustaining the fibrotic functions of BOS MCs. MNK signaling was upregulated in BOS cells and genetic or pharmacologic targeting of MNK activity inhibited eIF4E phospho-S209 and fibrotic functions of BOS MCs in vitro. In vivo treatment with MNK inhibitor eFT-508 was found to decrease allograft fibrosis in orthotopic murine lung transplant model. In Aim 1 we will utilize MCs derived from patients ± BOS to dissect the molecular mechanisms via which eIF4E Ser209 phosphorylation contributes to fibrogenic transformation of human lung MCs. Aim 2 will investigate the role of MNK/eIF4E signaling in development of BO in a murine orthotopic lung transplant model of BO by utilizing MNK1/2 Knockout and eIF4ES209A/A mutant mice to target MNK/eIF4E S209 in donor lungs. Preclinical studies of a novel orally available MNK1/2 inhibitor eFT508 in murine orthotopic whole lung transplant BO model with assessment of fibrotic and immune/inflammatory endpoints proposed in Aim 3 will lay the background for future human therapeutic trials. Together these studies will shed novel light on the role of MNK/eIF4E Ser209 phosphorylation in fibrotic activation of MCs and test its potential as an attractive target for halting fibrogenic progression and development of CLAD.