Deciphering the molecular pathogenesis of Cushing's disease

垂体腺瘤(库欣病，CD)自主分泌ACTH会导致肾上腺产生过量皮质醇，从而增加发病率和死亡率。这种罕见的内分泌疾病的治疗选择有限。即使在成功的神经外科手术后，复发率也很高，患者需要治疗。更好地了解导致CD的分子事件对于改进现有的治疗策略是必要的。2015年，我们已经确定了第一个主要的驱动突变，即激活脱泛素酶USP8的突变(在40%的CD中可检测到)。在之前的资助期间，我们首先描述了USP48的反复突变是另外10%的CD的驱动突变，使泛素系统的缺陷成为CD的主要原因。我们现在的目标是进一步阐明这些突变对CD生物学的影响。为此，我们的项目分为四个工作包：1)所有已发表的关于USP8的功能分析都是在唯一可用的促肾上腺皮质细胞系，小鼠垂体细胞系AtT-20中进行的，在USP8突变体过表达后进行。然而，这种细胞系有几个缺点，包括小鼠来源。利用已建立的从CD组织分离的原代细胞的方法学，我们旨在建立具有不同遗传背景的人CD细胞系，并将其用作2D和3D模型。2)我们的初步数据表明，突变体USP8和USP48解除了DNA损伤反应(DDR)和DNA修复途径的调控。我们将使用生化方法来评估蛋白质-蛋白质相互作用、蛋白质泛素化以及DDR信号和DNA修复的功能分析，来表征USP8和USP48突变对这些通路的影响。这些实验将在ATT-20细胞和来自人类肿瘤的细胞(WP 1)中进行。3)促肾上腺皮质激素通过增加皮质醇分泌而产生免疫抑制作用。我们的初步数据也提供了USP8突变诱导炎症过程的证据。在这个工作包中，我们将使用全面的免疫荧光分析和突变免疫原性的电子预测来更深入地分析免疫系统和CD肿瘤之间的这种双重相互作用。4)在这个翻译工作包中，我们将建立预后标志物和潜在的治疗靶点。通过对12个“侵袭性”CD肿瘤和12个“非常良性”CD肿瘤进行分类，我们将应用单细胞测序以及NanoString®的空间分子成像技术。通过这两种方法，我们将更多地了解CD肿瘤微环境的复杂性，但我们也将识别可作为生物标记物和/或可药物靶点的改变的途径。总体而言，我们相信我们的研究将提高我们对CD发病机制的理解，并将为开发更有效的治疗方法提供重要的见解。

Autonomous ACTH secretion of pituitary adenomas (Cushing's disease, CD) leads to cortisol overproduction by the adrenal glands and, thereby, to increased morbidity and mortality. Therapeutic options of this rare endocrine disease are limited. Even after successful neurosurgical resection, recurrence rate is high and patients require medical treatment. Better understanding of the molecular events that cause CD are necessary to improve the existing treatment strategies. In 2015, we have identified the first major driver mutations, namely activating mutations of the deubiquitinase USP8 (detectable in 40% of CD). In the previous funding period, we first described recurring mutations in USP48 as driver mutation in another 10% of CD making defects in the ubiquitin system the major cause of CD. We now aim to further elucidate the impact of these mutations on the biology of CD. For this purpose our project is divided in four work packages: 1) All published functional analyses on USP8 were performed in the only available corticotroph cell line, the murine pituitary cell line AtT-20, after overexpression of USP8 mutants. However, this cell line has several drawbacks, including the murine origin. Using the established methodology on primary cells dissociated from CD tissue, we aim at creating human CD cell lines with different genetic backgrounds and use them both as 2D and 3D models. 2) Our preliminary data suggest that mutant USP8 and USP48 deregulate DNA damage response (DDR) and DNA repair pathways. We will characterize the impact of USP8 and USP48 mutations on these pathways using biochemical methods to assess protein-protein interactions, protein ubiquitination as well as functional analysis of DDR signaling and DNA repair. The experiments will be performed both in the AtT-20 cells and the cells derived from human tumors (WP 1). 3) ACTH induces via increased cortisol secretion potent immune inhibitory effects. Our preliminary data provide also evidence of an induction of inflammatory processes by USP8 mutations. In this work package, we will analyze in more depth this dual interaction between the immune system and the CD tumors using comprehensive immunofluorescence analyses and in silico prediction of mutation immunogenicity. 4) In this translational work package, we will establish prognostic markers and potentially therapeutic targets. Using a very well characterized cohort of 12 'aggressive' vs. 12 'very benign' CD tumors, we will apply single cell sequencing as well as the spatial molecular imaging technique by Nanostring®. With these two methods, we will learn more about the complexity of the CD tumor microenvironment, but we will also identify altered pathways that can serve as biomarkers and/or 'drugable targets'. Overall, we are confident that our studies will improve our understanding of the pathogenesis of CD and will provide important insight for the development of more effective therapeutics.