Deciphering the molecular pathogenesis of Cushing's disease

垂体腺瘤（库欣病，CD）的自主ACTH分泌导致肾上腺皮质醇过量产生，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是有限的。即使在成功的神经外科切除后，复发率也很高，患者需要接受药物治疗。更好地了解导致乳糜泻的分子事件对于改进现有的治疗策略是必要的。2015年，我们确定了第一个主要的驱动突变，即去泛素酶USP8的激活突变（在40%的CD中可检测到）。在之前的资助期内，我们首先将USP48的反复突变描述为另外10%的CD的驱动突变，使泛素系统中的缺陷成为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皮质细胞系——小鼠垂体细胞系at -20中进行的，该细胞系在USP8突变体过表达后。然而，这种细胞系有几个缺点，包括小鼠起源。利用从乳糜泻组织分离的原代细胞的既定方法，我们的目标是创建具有不同遗传背景的人类乳糜泻细胞系，并将它们用作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。我们将描述USP8和USP48突变对这些途径的影响，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。实验将在AtT-20细胞和人类肿瘤细胞（WP 1）中进行。3)ACTH通过增加皮质醇分泌诱导强大的免疫抑制作用。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计算机预测更深入地分析免疫系统和CD肿瘤之间的双重相互作用。4)在这个转化工作包中，我们将建立预后标志物和潜在的治疗靶点。我们将使用Nanostring®的单细胞测序和空间分子成像技术，对12个“侵袭性”和12个“非常良性”的CD肿瘤进行很好地表征。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，但我们也将确定可以作为生物标志物和/或“可药物靶点”的改变途径。总的来说，我们相信我们的研究将提高我们对乳糜泻发病机制的理解，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.