Endocrine Abstracts

Introduction: Bilateral macronodular adrenocortical disease (BMAD) is an adrenal cause of Cushing’s syndrome explained in a subset of patients by inactivation of the tumor suppressor genes ARMC5 or KDM1A. Genetic and transcriptomic studies cluster BMAD in 3 distinct molecular groups: one with ARMC5 inactivation, one with KDM1A inactivation and a third group with no known genetic cause. Although ARMC5 and KDM1A are involved in the post-translational regulation of proteins, the protein studies in BMAD tissues are mainly limited to immunohistochemistry targeted on steroidogenesis enzymes. The aim of this work was to study the proteomic heterogeneity in a BMAD cohort and its correlation with the molecular causes.

Patients and Methods: The cohort includes 24 BMAD (7 ARMC5-altered, 4 KDM1A-altered and 13 of unknown genetic cause) and 12 samples used as controls (10 adenomas and 2 normal adrenal glands). All patients underwent surgery in our center between 2006 and 2021. Samples were obtained from formalin-fixed and paraffin-embedded material by macrodissection. After protein extraction, identification and protein quantifications were performed by Light chromatography and mass spectrometry (LC-MS/MS) on the Cochin Institute Proteomic platform.

Results: PCA showed a rather homogeneity of ARMC5 and KDM1A-altered BMAD, whereas BMAD with no known genetic cause were more heterogeneous. In accordance with ARMC5 role in the regulation of the RNA polymerase II, several of its subunits including POLR2A are the most overexpressed proteins in ARMC5-mutated BMAD compared to the others BMAD groups. However, they have a low expression of several cholesterol biosynthesis enzymes (such as FDFT1 or NSDHL) compared to other BMAD. KDM1A-altered BMAD were also a homogeneous group characterized by the systematic loss of KDM1A protein and the overexpression of proteins involved in lipid metabolism (such as LDLR or SULT2A1).

Discussion: Our study is the first to explore the BMAD proteome using LC-MS/MS. We confirmed the high expression of ARN polymerase II subunits in ARMC5-altered BMAD and showed a high expression of ARN polymerase II partners smggesting its important role in the pathogenesis of BMAD mediated by ARMC5 alterations. Compared to other BMAD, ARMC5-altered BMAD expressed lower levels of cholesterol biosynthesis enzymes that could partially explain the low cortisol production of cells with bi-allelic inactivation of ARMC5. KDM1A altered BMAD showed an overexpression of proteins involved in lipid metabolism that probably play a role in the production of cortisol dysregulation. BMAD with no known genetic cause showed various proteomic pattern smggesting different pathogenic mechanisms to be identified.