Endocrine Abstracts

Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor resulting in excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases USP8 and USP48, and in BRAF genes, have been reported in a subset of patients affected by CD. Aim of this study was to characterize the genetic profile of a cohort of 66 patients with ACTH-secreting tumors, searching for somatic mutations in USP8, USP48 and BRAF hotspot regions. 7 patients were found to carry USP8 somatic mutations in the 14-3-3 protein binding motif (n = 5 P720R, n = 1 P720Q, n = 1 S718del); 2 patients were mutated in USP48 (M415I); no mutation was identified in BRAF. In addition, a novel USP8 variant, G664R, located in exon 14, upstream the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of G664R USP8variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the G664R USP8 variant resulted in a significant increase of ACTH release (201.1 ± 63.7% vs empty vector transfected cells, P <0.05) and of cell proliferation (141.8±30.5% vs empty vector transfected cells, P <0.05). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs WT-USP8, P <0.05). Surprisingly, In situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8-14.3.3 proteins colocalization, in USP8 G664R transfected cells with respect to USP8 WT transfected cells (-47.9±6.6%, vs WT-USP8, P <0.001), thus suggesting a possible conformational rearrangement due to amino acid 664 replacement affecting the binding of USP8 with partner proteins. No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage were observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT-USP8 and other USP8 mutants, G664R USP8 display an exclusive cytoplasmic localization. In conclusion, recurrent somatic mutations were found in USP8 (10.6% vs 36.5% incidence of all published mutations) and in USP48 (3% vs 13.3% incidence) hot spot regions. A novel USP8 variant was identified in a CD patient. In vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis.