Comprehensive Proteomic Classifier for the Molecular Characterization of Pulmonary Sarcoidosis

项目总结 本研究的主要目标是构建肺结节病的预测模型(分类器)和 进展性(P)与非进展性(NP)疾病，最终将有助于改善肺部疾病的预后 结节病。我们组建了一支独特的调查团队，拥有蛋白质组学、免疫学、 基因组学、结节病临床护理以及生物信息学和统计学。结节病是一种诊断性 挑战免疫介导的系统性疾病。它会导致严重的发病率和死亡率，主要原因是 进行性肺部疾病，尽管导致肺部疾病和P与NP疾病的因素是 未知。治疗肺结节病的策略，包括启动治疗的触发因素，是非 特效性；治疗通常依赖于用皮质类固醇抑制免疫系统，并与 相当大的副作用。肺和血液中的转录变化已确定为P病的特征 在横断面研究中。由于蛋白质是细胞功能的主要效应者，它们的变化导致 生物系统的破坏和疾病的发展，它们是生物标记物的合理来源。我们的预赛 来自支气管肺泡灌洗液和细胞的数据显示，肺组织中蛋白质组有显著的变化 结节病与对照组和P与NP病。我们假设有效的疾病标志物和那些 区分进展性和非进展性疾病将反映活跃在 疾病和进展。其次，通过表征细胞蛋白质、全球磷酸化事件和 细胞特异性RNA表达，我们将定义已知的蛋白质/基因/通路，如PI3K/Akt/mTOR和 其他丝氨酸-苏氨酸激酶信号转导机制以及新的致病蛋白/基因，如 胞内和芳香烃受体的信号转导，这将对机制和治疗有意义。我们 将使用高分辨率质谱学(MS)、先进的生物信息学和计算工具 表型结节病患者。在目标1中，我们将确定用于诊断的疾病特定分类器 使用Discovery结节病病例队列和患病和健康对照(已招募)的结节病 用于结节病病例和对照的开发和验证队列(为本研究招募)以验证 并对分类器性能进行了优化。在目标2中，我们将使用以下方法识别P和NP疾病的蛋白质分类器 与目标2相同的方法。在目标3中，我们将使用一种新的单细胞RNA测序方法，CITE-SEQ 识别特定细胞的转录，并将其与蛋白质变化相结合，包括检查全局 识别激酶信号和发现相关的细胞特异性细胞蛋白/基因的磷酸化事件 在我们的验证队列的子集中有疾病和进展。在本研究结束时，我们将定义 疾病和进展的诊断生物标记物，可以很容易地翻译到临床上。我们也将从中获益 对可能作为潜在治疗靶点的结节病肺蛋白和转录本的洞察 为今后的研究提供潜在的机理信息。

PROJECT SUMMARY The primary goal of this study is to construct predictive models (classifiers) of pulmonary sarcoidosis and progressive (P) vs. non-progressive (NP) disease that will ultimately serve to improve outcomes of pulmonary sarcoidosis. We have assembled a unique investigative team with expertise in proteomics, immunology, genomics, sarcoidosis clinical care, as well as bioinformatics and statistics. Sarcoidosis is a diagnostically challenging immune-mediated systemic disease. It results in significant morbidity and mortality, primarily due to progressive pulmonary disease, although the factors that drive pulmonary disease and P vs. NP disease are unknown. The strategies to treat pulmonary sarcoidosis, including the triggers to initiate treatment, are non- specific; treatment usually relies on suppressing the immune system with corticosteroids and is associated with considerable side-effects. Transcriptional changes in the lung and blood have defined a signature of P disease in cross-sectional studies. Since proteins are the main effectors of cellular function and their alterations result in disruption of biologic systems and disease development, they are a logical source of biomarkers. Our preliminary data from bronchoalveolar lavage fluid and cells demonstrate significant proteome wide alterations in pulmonary sarcoidosis vs controls and P vs NP disease. We hypothesize that effective markers of disease and those distinguishing progressive from non-progressive disease will reflect biological processes active in disease and progression. Secondarily, by characterizing cellular proteins, global phosphorylation events and cell-specific RNA expression, we will define known proteins/gene/pathways such as the PI3K/Akt/mTOR and other serine-threonine kinase signaling mechanisms as well as novel pathogenic proteins/genes, such as endocytic and aryl hydrocarbon receptor signaling, which will have implications for mechanism and therapy. We will use high-resolution mass spectrometry (MS), advanced bioinformatics and computational tools in well- phenotyped sarcoidosis patients. In Aim 1, we will determine a disease-specific classifier for diagnosing sarcoidosis using a Discovery Cohort of sarcoidosis cases and diseased and healthy controls (already recruited) for the development and Validation Cohort (recruited for this study) of sarcoidosis cases and controls to verify and optimize the classifier performance. In Aim 2, we will identify a protein classifier of P vs NP disease using the same approach as in Aim 2. In Aim 3 we will use a novel single-cell RNA-sequencing approach, CITE-seq to identify transcription from specific cells, and integrate it with protein changes, including examination of global phosphorylation events to identify kinase signaling and discover cell-specific cellular proteins/genes associated with disease and progression in a subset of our Validation Cohort. At the end of this study, we will have defined diagnostic biomarkers of disease and progression that can be translated easily to the clinic. We will also gain insights into the sarcoidosis pulmonary proteins and transcripts that may serve as potential therapeutic targets and provide potential mechanistic information with future study.