MON-468 Selective Inhibition of Epigenetic Pathways Has Anti-Proliferative and Pro-Apoptotic Effects on the Mouse Corticotroph Tumor Cells, AtT20

Abstract

Corticotrophinomas, which are neuroendocrine tumors (NETs) and represent >10% of all surgically removed pituitary adenomas, cause Cushing’s disease that is associated with hypersecretion of adrenocorticotropic hormone (ACTH) leading to excessive production of glucocorticoids by the adrenal cortex. Trans-sphenoidal hypophysectomy is the treatment of choice for corticotrophinomas, with medical treatments being reserved for patients who have contraindications for surgery. However, these treatments are often ineffective. Improved therapeutic approaches are required, and we have therefore assessed the efficacy of epigenetic modulators, a new class of anti-cancer drugs that have been reported to be effective in treating pancreatic NETs. Specifically, we assessed the anti-proliferative and pro-apoptotic effects of the bromo and extra terminal domain (BET) inhibitors JQ1 and PFI-1, in the mouse corticotrophinoma cell line AtT20, using Cell Titer Blue and Caspase Glo assays, respectively. JQ1, after 96h treatment, was more efficacious than PFI-1. Thus, JQ1 significantly decreased proliferation by 95%, (p<0.0005), and significantly increased apoptosis >50-fold (p<0.0005), compared to control treated cells; whereas PFI-1 decreased proliferation by 43% (p<0.0005), but did not significantly alter apoptosis. Furthermore, AtT20 cells did not resume proliferating for up to 96 hours after the removal of JQ1 from the media. In addition, RNA-sequence (RNA-Seq) analysis revealed that JQ1 treatment significantly altered the expression of genes involved in apoptosis, including Nuclear Factor Kappa B Subunit 1 (Nfκb1) and Baculoviral IAP Repeat Containing 3 (Birc3), as well as genes associated with the somatostatin receptor type 2 (SSTR2) anti-proliferative signaling pathway, including Sstr2. The down regulation of these genes was confirmed using quantitative PCR, which showed decreases in Nfkb1, Birc3 and Sstr2 mRNA of 2.9-fold (p<0.0009), 1.7-fold (p<0.005), and 1.5-fold (p<0.005), respectively. In addition, Western blot analysis showed decreases in Nfkb1, cIAP2 and SSTR2 protein expression of 1.5-fold (p<0.05), 1.9-fold (p<0.05), and 2.3-fold (p<0.05), respectively. Thus our results, which demonstrate that JQ1 treatment is both anti-proliferative and pro-apoptotic in ACTH-secreting cells, reveal that BET inhibition may provide a novel approach for the treatment of corticotrophinomas.