OR25-04 Methylation Status of the POMC Promoter in Human ACTH-secreting Pituitary Tumors: Validation of Glucocorticoid-induced Methylation Patterns Based on Clinical Phenotype

Abstract Disclosure: M. Usutani: None. S. Takayasu: None. S. Yamada: None. Y. Fujita: None. Background: Cushing disease is caused by the dysregulation of ACTH synthesis and secretion due to corticotroph tumors (ACTHomas). Typical ACTHomas exhibit a partial lack of response to glucocorticoid (GC) negative feedback, although the underlying mechanism remains largely unclear. Recently, a second POMC promoter regulated by methylation status was identified in human ACTHomas. However, it is currently unknown whether the methylation levels of the POMC promoters are involved in the GC resistance observed in ACTHomas. Method: To assess methylation/demethylation positions in the first and second promoters, we performed bisulfite-conversion-based DNA sequencing of the CpG islands in 16 ACTHoma cases. The expression levels of ten-eleven translocation (TET) enzymes and DNA methyltransferases (DNMT) were evaluated using qPCR. The same experiments were conducted using the murine corticotroph tumor cell line AtT-20. Subsequently, we investigated changes in the methylation status of the two POMC promoters in human primary cultured cells following treatment with synthetic GC Dexamethasone (DEX), and explored their association with clinical GC resistance. Results: For the first promoter, we found that in the group where methylation decreased after DEX treatment, basal cortisol levels and cortisol levels after low-dose DEX administration were significantly higher compared to the group with increased methylation. No correlation was found between methylation levels and clinical data for the second promoter. The expression levels of TET and DNMT families were significantly increased by DEX in the group where methylation of the first promoter increased after DEX treatment. In contrast, the CpG islands of the first and second promoters were highly demethylated in AtT-20 cells, and DEX treatment had no effect on their methylation levels. The expression levels of Tet and Dnmt were slightly decreased following DEX treatment in AtT-20 cells. Discussion: The decrease in methylation of the first promoter appears to correlate with an increase in basal cortisol levels and cortisol levels after the low-dose DEX test, suggesting that GC-induced demethylation in the first promoter may partially contribute to resistance to the negative regulation of POMC transcription in ACTHomas. Although DEX treatment did not alter methylation levels in the second promoter, it was confirmed that basal cortisol levels were higher in the group with low basal methylation levels in the second promoter. Thus, the methylation status of the second promoter may regulate basal cortisol levels. Furthermore, differences between species and samples should be considered, as these may affect the results. Conclusion: Low methylation of the second POMC promoter was found to increase serum cortisol levels, and the degree of resistance to GCs may be regulated by GC-induced demethylation levels of the first POMC promoter in ACTHomas. Presentation: Monday, July 14, 2025