Uterine signaling networks in the pathogenesis of pulmonary lymphangioleiomyomatosis (LAM)

项目总结 淋巴管肌瘤病(LAM)是一种影响育龄妇女的肺部肿瘤，其原因是- 结节性硬化症1(TSC1)或TSC2基因功能突变。机械性靶点的过度激活 雷帕霉素1导致囊性肺重塑和进行性呼吸衰竭。西罗莫司治疗稳定肺功能 LAM患者的功能和改善症状，但长期的益处和毒性尚不清楚，一些 患者对治疗没有反应。LAM细胞是转移性的，但原始组织来源不明。 LAM肺部特异性转移、原发发病机制和女性患病率不明 是开发新的治疗方法的障碍。我们对LAM肺单个细胞的初步数据 RNA测序(ScRNAseq)分析确定了一组独特的细胞(LAMCORE)表达子宫- 在正常肺中未检测到的特定同源框(HOX)转录因子(TF)；LAM的TF谱 患者来源的细胞证实激活了前B细胞白血病同源盒1(Pbx1)，这是HOXs的辅助因子； LAM肺癌scRNAseq和乳腺癌细胞Pbx1-ChIPseq的比较分析显示过度表达 具有阳性Pbx1结合峰的LAMCORE特征基因。总而言之，这强烈表明起源于子宫。 HOX/Pbx1信号通路在LAM发病机制中的中心作用。这项提议的目标是 探讨HOXs相关基因网络调控LAM发病和进展的机制。 我们的中心假设是Hox/Pbx1在女性下游协调了一个细胞特异的基因网络 调节LAMCORE细胞存活和肺转移的激素。提出了三个具体目标来 HOX/Pbx1基因网络参与LAM损伤的分子机制 转移、形成和发展。目的1：确定肺LAM特异性基因组和表观基因组 HOX/Pbx1介导LAM发病的反应和机制。目标2：确定 Hox/Pbx1网络基因在体内外对LAM细胞转移潜能的功能影响 目的3：确定HOX/Pbx1拮抗剂HXR9单独或与西罗莫司合用对血管紧张素转换酶活性的影响 雌激素促进体内肺转移潜能和肺重塑。完成这项提案 将首次提供：1)LAMCORE的高分辨率一体化基因组/表观基因组蓝图 细胞，2)LAMCORE细胞特异性Hox/Pbx1基因网络，其中包含预测的关键调控因子和靶点 随访扰动和临床诊断测试的网络，3)对Hox/Pbx1的新机制见解 调节回路及其对LAM发病机制的功能影响，以及4)临床前原则证据 靶向Hox/Pbx1调节的女性激素介导的LAM进展作为一种新的缓解诱导 LAM患者的治疗策略。我们的工作对于理解LAM具有重大的生物医学意义 发病机制，发展LAM诊断新方法和设计替代缓解诱导 治疗策略，以最大限度地提高LAM治疗的益处/毒性比。

PROJECT SUMMARY Lymphangioleiomyomatosis (LAM), a lung neoplasm affecting reproductive age women, is caused by loss-of- function mutations in tuberous sclerosis 1 (TSC1) or TSC2 genes. Hyperactivation of the mechanistic target of rapamycin 1 leads to cystic lung remodeling and progressive respiratory failure. Sirolimus therapy stabilizes lung function and improves symptoms in LAM patients, but the long-term benefit and toxicity are unknown, and some patients do not respond to therapy. LAM cells are metastatic but the primary tissue of origin is unknown. Unknown mechanisms underlying lung-specific metastasis, primary pathogenesis and female prevalence of LAM are obstacles to the development of new curative therapies. Our preliminary data from LAM lung single cell RNA sequencing (scRNAseq) analysis identified a unique population of cells (LAMCORE) expressing uterine- specific homeobox (HOX) transcription factors (TFs) that are not detected in normal lung; TF profiling of LAM patient-derived cells identified activation of Pre-B-cell leukemia homeobox 1 (PBX1), a cofactor of HOXs; comparative analysis of LAM lung scRNAseq and breast cancer cell PBX1-ChIPseq revealed overly represented LAMCORE signature genes with positive PBX1 binding peaks. Collectively, this strongly suggests a uterine origin of LAM cells and a central role of HOX/PBX1 signaling in LAM pathogenesis. The objective of this proposal is to identify mechanisms by which HOXs-associated gene networks regulate LAM pathogenesis and progression. Our central hypothesis is that HOX/PBX1 orchestrates a cell-specific gene network downstream of female hormones that regulates the survival and lung metastasis of LAMCORE cells. Three specific aims are proposed to delineate the molecular mechanisms through which HOX/PBX1 gene network contributes to LAM lesion metastasis, formation and progression. Aim 1: Determine pulmonary LAM-specific genomic and epigenomic responses and mechanisms underlying HOX/PBX1-mediated LAM pathogenesis. Aim 2: Determine the functional impacts of HOX/PBX1 network genes on metastatic potentials of LAM-derived cells in vitro and in vivo. Aim 3: Determine the effect of the HOX/PBX1 antagonist, HXR9, singly or in combination with Sirolimus, on estrogen-promoted lung metastasis potentials and lung remodeling in vivo. The completion of this proposal will provide for the first time: 1) a high-resolution integrative genomic/epigenomic blueprint of the LAMCORE cells, 2) a LAMCORE cell-specific HOX/PBX1 gene network with predicted key regulatory factors and targets within the network for follow-up perturbation and clinical diagnostic tests, 3) novel mechanistic insights into HOX/PBX1 regulatory circuits and their functional impact on LAM pathogenesis, and 4) preclinical proof-of-principle evidence for targeting HOX/PBX1-regulated female hormone-mediated LAM progression as a novel remission-inducing therapeutic strategy for LAM patients. Our work has major biomedical relevance for understanding LAM pathogenesis, developing new approaches for LAM diagnosis and designing alternative remission-inducing therapeutic strategies to maximize the benefit/toxicity ratio LAM treatment.