OR01-02 Epigenomic Dysregulation Of PPP1R17 Drives The Cell Cycle In Adenomas Causing Cushing’s Disease
Authors: Asuzu David , Mullaney Dustin , Stoica Stefan , Mandal Debjani , Maric Dragan , Elkahloun Abdel , Sisay Bayu , Tatsi Christina , Nieman Lynnette K , Chittiboina Prashant 
Published: October 2023

Disclosure: D. Asuzu: None. D. Mullaney: None. S. Stoica: None. D. Mandal: None. D. Maric: None. A. Elkahloun: None. B. Sisay: None. C. Tatsi: None. L.K. Nieman: None. P. Chittiboina: None.

Introduction: Pituitary adenomas causing Cushing’s disease are largely (60%) mutationally bland. Previously, with syngeneic, pairwise transcriptomic analysis, we found a robust upregulation of PPP1R17 in canonically mutated (USP8, USP48 and BRAF) and ‘wildtype’ CD adenomas. PPP1R17 is an endogenous inhibitor of tumor suppressor phosphatase PP2A. Here, we investigated the epigenomic dysregulation of PPP1R17, its interaction with PP2A, and downstream effects on cell cycle regulation. Methods: We performed single nucleus assays for transposase accessible chromatin (snATACseq, 10X Genomics) in adenomas from 4 CD patients, and two syngeneic adenoma-normal pairs along with parallel single nucleus RNAseq (snRNAseq). Transcription factors bound to open chromatin were identified by reverse chromatin immunoprecipitation (rChIP) using CRISPR dCas9-3XFLAG-Biotin (Millipore Sigma) and promoter-specific synthetic guide RNAs (Invitrogen TrueGuide). Bound transcription factors were quantified using mass spectrometry (SimulTOF 300). Fixed human CD adenomas (n=5) were sectioned and stained using multiplexed immunohistochemistry (mIHC). Association between R17 and protein phosphatase 2A (PP2A) was assessed using proximity ligation assays (PLA, Sigma). We overexpressed R17 or GFP controls in murine corticotroph (mCort) cell lines and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo). Results: We mapped the chromatin accessibility landscape of CD adenomas compared to normal corticotrophs. We identified coordinated chromatin accessibility (snATACseq) and transcriptional upregulation (snRNAseq) at the R17 locus exclusively within adenomatous corticotrophs. Using rChIP, we identified enrichment at the R17 promoter of the RNA polymerase II transcriptional coactivator p15 (SUB1) and the histone variant H1.4, indicating active chromatin remodeling. PPP1R17 protein and its target PP2A were overexpressed in the adenoma compartment but not in the adjacent normal gland. PLA confirmed robust interaction between R17 and PP2Ac within the CD adenoma compartment. Mechanistically, mCortR17 cells showed inhibition of PP2A with phosphorylation its known targets - EGFR and ERK. mCortR17 cells showed accelerated cell cycle progression compared to mCortGFP (S-phase 16.4% versus 24.1%, P &lt; 0.001) with increased proliferation (two-way ANOVA P &lt; 0.001). PP2A agonists Fingolimod, DT061 and ABL127 led to proliferation arrest in mCortR17 cells. Conclusions: We identified increased R17 expression as an underlying mechanism of tumor hyperproliferation in CD adenomas. We found chromatin accessibility underlying PPP1R17 overexpression. PP2A agonists reversed the effects of PPP1R17 overexpression in-vitro. Our findings uncover a novel therapeutic phosphatase target for patients with CD.

Presentation: Thursday, June 15, 2023