THU478 Unchecked Kinase Activation Underlies Pituitary Adenomas Causing Cushing’s Disease

Disclosure: D. Asuzu: None. D. Mullaney: None. Y. Li: None. D. Mandal: None. C. Tatsi: None. L.K. Nieman: None. P. Chittiboina: None. Introduction: Common mechanisms underlying canonically mutated (USP8, USP48 and BRAF) and ‘wildtype’ pituitary adenomas causing Cushing’s disease remain unknown. Using syngeneic, pairwise transcriptomic analysis at the single cell/nucleus resolution, we found a ubiquitous and robust overexpression of PPP1R17 in CD adenoma corticotrophs. The role of PPP1R17, an endogenous inhibitor of tumor-suppressor phosphatase PP2A in the embryonic context remains unknown in the pituitary. Here, we elucidated mechanistically, the effect of PPP1R17 on the phopshoproteome of CD adenomas. Methods: We quantitatively profiled the proteome and phosphoproteome of three pairs of CD adenomas and their syngeneic adjacent normal pituitary glands with TMT labeling and mass spectrometry (LC-MS/MS). We validated the mechanisms with murine corticotroph cells (mCort) overexpressing R17 (mCort(R17)) or GFP (mCort(GFP)). Elevated total and phosphprotein expression was assessed using R packages by performing cross sample normalization, noise filtering and differential protein abundance or phosphorylation analyses. Pathway enrichment analysis was performed on the KEGG database using ShinyGo 0.76.2. Results: We identified 37 proteins differentially phosphorylated in CD adenomas compared to adjacent normal pituitary glands, including the AP2-associated protein kinase 1 (Aak1) and the apoptotic chromatin condensation inducer Acin1. Of these 37 proteins, 30 (81%) were also differentially phosphorylated in mCort(R17) versus mCort(GFP) cells, suggesting that differential phosphorylation in CD is driven by R17 hyperactivity. Of the 35 proteins overabundant in CD adenomas, 15 (43%) overlapped with mCort(R17), including the Myc-associated protein (Max) and Presenilin 1 (Psen1). Proteins enriched in both human CD and mCort datasets were involved in NOTCH signaling and neurotransmitter release, while phospho-proteome profiling highlighted R17-mediated perturbation of RNA polymerase II transcription termination and mRNA processing. Conclusions:We first discovered a heavily dysregulated phosphoproteome in CD adenomas compared to syngeneic normal glands. We found that we could replicate the signature phosphoproteomic profile by overexpressing PPP1R17 in-vitro. These data indicate a central role for phosphatase (PP2A) inhibition in CD adenomas, and suggest potential novel anti-tumor targets for patients with CD. Presentation: Thursday, June 15, 2023