Introduction: Recently, our group demonstrated that SAHA, a histone deacetylase inhibitor, provides a potential novel strategy for recurrent/unremitting Cushing’s disease (CD) via anti-tumor/hormonal effects.[1] Interestingly, SAHA differentially reduced transcription of proopiomelanocortin (POMC) in adenomatous, but not normal corticotrophs. Here, we elucidate the mechanism that could underlie such a differential action of SAHA, and potentially provide new therapeutic avenues to control hormone secretion.
Methods: ACTH secretion (ELISA), apoptosis (FACS/RT-qPCR/immunoblot), and gene expression profile (microarray) was examined in AtT-20 cells with or without SAHA (0 – 4 µM). In-vivo efficacy of SAHA (50 mg/Kg) was examined in NCR-nude mouse AtT-20 xenograft model. SAHA’s efficacy against human-derived CtT (hCtT) (n=7) was assessed in-vitro. The link between POMC transcriptional regulation and SAHA was investigated using CRISPR/dCas9 activation system[2] in AtT-20 cells.
Results: Consistent with clinical observations[3], we confirmed that short term exposure (3h) or longer term (5d) exposure to SAHA (50mg/Kg), did not result in ACTH reduction in control animals. Independent of apoptosis mediated cytotoxicity of SAHA[1,4], we detected an early (3h) reduction in AtT20 ACTH secretion in-vitro (70%,p<0.0001) and in-vivo (76%, serum ELISA). We detected diminished POMC transcription (39%,p=0.0001) and reduced POMC promoter activation. Microarray analysis revealed liver X receptor alpha (LXR) and POMC downregulation with SAHA. Accordingly, SAHA reduced LXR in AtT-20 but, not in normal murine corticotrophs while its obligate heterodimer retinoid X receptor alpha (RXR)[5], was unaffected. Finally, to confirm LXR mediated effect on POMC , LXR transcriptional augmentation was achieved using CRISPR/dCas9. The resultant overexpression rescued AtT-20 cells from SAHA-mediated (1-4µM/3h) POMC downregulation and ACTH secretion.
Conclusions: Our findings elucidate the mechanisms underlying the differential effect of SAHA in reducing ACTH secretion in adenomatous cells. Normal corticotrophs escape this effect likely via LXR, making SAHA a promising drug for treatment of recurrent/residual CD.
Patient Care: Our study has provided mechanistic insight into how the FDA approved, oral HDACi – SAHA has anti-hormonal activity in corticotroph tumors. These novel findings could allow the discovery of more targeted pharmacotherapies in the management of recurrent/unremitting Cushing’s disease.
Learning Objectives: By the conclusion of this session, participants should be able to:
1) Describe the importance of need to devise effective therapies for biochemical remission in recurrent or unremitting Cushing’s disease.
2) Discuss the effects of SAHA on LXR and the consequential POMC transcriptional downregulation observed in-vivo and in-vitro.
3) Identify a novel pharmacological target in the treatment for recurrent/unremitting Cushing’s disease with SAHA.
References: 1. Chittiboina P, Lu J, Wang X, Piazza MG, Zhuang Z. 213 Histone Deacetylase Inhibitor Vorinostat Is a Novel, Promising Treatment for Cushing Disease. Neurosurgery. 2016;63 Suppl 1:183. doi:10.1227/01.neu.0000489782.99169.41.
2. Konermann S, Brigham MD, Trevino AE, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2014;517(7536):583-588. doi:10.1038/nature14136.
3. Mann BS, Johnson JR, Cohen MH, Justice R, Pazdur R. FDA approval summary: vorinostat for treatment of advanced primary cutaneous T-cell lymphoma. Oncologist. 2007;12(10):1247-1252. doi:10.1634/theoncologist.12-10-1247.
4. Sangeetha SR, Singh N, Vender JR, Dhandapani KM. Suberoylanilide hydroxamic acid (SAHA) induces growth arrest and apoptosis in pituitary adenoma cells. Endocrine. 2009;35(3):389-396. doi:10.1007/s12020-009-9159-1.
5. Hong C, Tontonoz P. Liver X receptors in lipid metabolism: opportunities for drug discovery. Nat Rev Drug Discov. 2014;13(6):433-444. doi:10.1038/nrd4280.