DDDR-22. Fingolimod targets Cushing’s disease adenomas in-vitro and in-vivo

Abstract INRODUCTION Cushing’s disease (CD) causes severe morbidity and up to 50% higher mortality despite best medical practices. Effective targeted therapies are lacking for CD. Our objective was to identify and test novel agents targeting CD. METHODS We performed single nucleus RNAseq (snRNAseq, 10X Genomics) on 4 CD adenomas including two syngeneic adenoma-normal pairs. We also performed DNA methylation (Illumina Infinium) on 5 CD adenomas compared to 11 autopsy-derived control pituitary glands. We treated ATT20 murine cells, a model of CD, with 5uM fingolimod (n = 8/group) and assessed proliferation (Promega OneGlo). We injected 5 million ATT20 cells into the flanks of athymic Nu/Nu mice. After allowing tumors to engraft, animals were randomized to fingolimod 1mg/Kg versus normal saline controls (n = 5/group). After 17 days, animals were sacrificed and tumor weights and volumes were measured. RESULTS snRNAseq and DNA methylation studies identified promoter hypomethylation and transcriptional upregulation of PPP1R17, an endogenous PP2A inhibitor. We therefore tested fingolimod, a potent PP2A agonist as a novel therapeutic agent for CD. Stable PPP1R17 overexpression in ATT20 cells led to hyperproliferation compared to ATT20GFP cells, and this was reversed by 5uM fingolimod. Fingolimod also induced proliferative arrest in ATT20GFP cells within 3 days (P < 0.05). In Nu/Nu mice bearing ATT20 xenografts, fingolimod 1mg/Kg decreased tumor weights (100 vs 249mg, P = 0.05) and tumor volumes (168 vs 463 mm3, P = 0.05). CONCLUSIONS PP2A inhibition via PPP1R17 overexpression is a targetable mechanism of CD tumorigenesis. The PP2A agonist fingolimod effectively targeted ATT20 cells both in-vitro and in-vivo. Our results highlight a novel therapeutic strategy for patients with CD.