Deciphering the molecular pathogenesis of Cushing's disease

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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.