Pituitary Adenomas Evade Apoptosis via Noxa Deregulation in Cushing's Disease

Introduction: Sporadic pituitary adenomas are common (>10%) in the general population. Microadenomas (< 1 cm across) can become symptomatic with adrenocorticotropin oversecretion causing Cushing’s disease (CD), leading to high morbidity and mortility. Pathogenic mechanisms in a majority of adenomas including wildtype CD adenomas remain unknown. Mechanistic studies are hindered by a lack of in-vitro models and normal human pituitary gland controls. We hypothesized that comparative analysis of human adenomas with syngeneic normal pituitary glands could reveal pathogenic mechanisms driving CD.   Methods: We surgically annotated adenomas and en-route adjacent normal glands in 34 patients. We performed single cell RNAseq and captured 28,724 cells in 6 patients (3 CD and 3 non-CD; 5 pairwise), bulk RNAseq in 7 patients (5 CD and 2 non-CD; 5 pairwise) and DNA methylation analysis in 6 patients (3 CD and 3 non-CD). We confirmed the mechanisms of sporadic CD adenoma pathogenesis in 2 additional syngeneic pairs of human cell lines and using a novel murine normal corticotroph-enriched cell line.    Results: Using marker-based classification, we confirmed canonical resident cell classes including corticotrophs in the human pituitary gland. We found SOX2+/S100B+ folliculostellate cells which play a role in pituitary cell replenishment and homeostasis in the normal gland but not in the adenoma compartment. Within the terminally differentiated corticotrophs in the adenoma compartment, we discovered a subset of proliferating cells with upregulation of canonical oncogenes, histone modifiers and DNA repair genes. We also found that consensus dominant genes including PMAIP1 (encoding the pro-apoptotic BH3 only Bcl-2 protein noxa) sharply demarcated adenoma cells from normal gland. The PMAIP1 locus was hypomethylated in CD adenomas compared to non-CD samples. However, despite epigenetic and transcriptional upregulation, we found proteasomal degradation of noxa protein in CD adenomas. Using the selective proteasomal inhibitor bortezomib, we were able to rescue noxa ex-vivo and in-vitro, induce apoptosis, and reduce cell survival of adenoma cell lines.  Conclusions: We defined the transcriptionl and DNA methylation landscape of human pituitary adenomas compared to adjacent normal gland tissue. We identified proteasomal-mediated noxa degradation despite PMAIP1 overexpression as an escape mechanism from apoptosis in adenomatous corticotrophs. The proteasomal inhibitor bortezomib rescued noxa and induced apoptosis in adenoma cells, highlighting its potential as a novel therapeutic strategy in CD.