198 Polycombs Drive Tumorigenesis in Aggressive Cushing’s Disease Adenomas

INTRODUCTION: The mechanism of progression of Cushing’s disease (CD) adenomas to an aggressive phenotype (Ki67 > 3%) remains unknown. Polycombs are master epigenetic regulators that catalyze H3K27 trimethylation and induce gene silencing in aggressive tumors. METHODS: We performed multiome analysis (single nucleus RNA-seq and ATAC-seq, 10x Genomics) in 5 aggressive CD adenomas, and verified key protein pathways using mIHC. Polycomb activity was assessed using H3K27me3 ChIP-seq. ATT20 cells were treated using polycomb inhibitors DZNep and Tazemetostat, HDAC inhibitor Vorinostat, or siRNA targeting polycombs Ezh2 and Suz12. We treated one patient off-label with oral Vorinostat 400mg daily for 6 days prior to radiation, and initiated a phase 0 clinical trial of Vorinostat in aggressive CD adenomas (CTN20N0019). RESULTS: Aggressive CD adenomas showed elevated EZH2 and EED chromatin accessibility. snRNA-seq confirmed robust EZH2, SUZ12 and EED overexpression, and mIHC showed abundance of SUZ12, EZH2 and H3K27me3 in aggressive CD adenomas. ChIP-seq in aggressive versus typical CD adenomas identified > 6,500 peaks with differential H3K27me3 occupancy. In ATT20 cells, treatment with DZNep, Vorinostat, siEzh2 or siSuz12 led to marked proliferative arrest (P < 0.05). Pomc overexpression was reversible using DZNep (FC 0.74, P = 0.03) or Vorinostat (FC 0.24, P = 0.02). In a patient with an aggressive CD adenoma, a single 6-day course of Vorinostat prior to external beam radiation treatment induced profound decline in serum cortisol (24 mcg/dL to 7.5 mcg/dL) and ACTH (63.7 pg/mL to 44.9 pg/mL), as well as radiographic tumor shrinkage. CONCLUSIONS: Polycomb overactivity drives aggressive CD adenomas. Polycomb and HDAC inhibitors reversed hyperproliferation and Pomc overexpression in-vitro, and HDAC inhibition showed therapeutic potential in a first-in-human application. Our studies uncovered a novel targetable mechanism of tumorigenesis in aggressive CD adenomas.