Abstract
Disclosure: N. Yamamoto: None. Y. Tsujimoto: None. H. Shichi: None. S. Urai: None. M. Suzuki: None. H. Bando: None. M. Yamamoto: None. Y. Motomura: None. Y. Sasaki: None. Y. Oi-Yo: None. M. Takahashi: None. G. Iguchi: None. D. Motooka: None. N. Fukuhara: None. H. Nishioka: None. S. Yamada: None. W. Ogawa: None. H. Fukuoka: None. Introduction: N6-methyladenosine (m(6)A) is the most abundant post-transcriptional modification in eukaryotic mRNA and is associated with various physiological mechanisms, including stress responses, through the regulation of RNA stability and translation efficiency. In patients with depression, a disturbance in the regulation of m(6)A by glucocorticoids has been suggested. Therefore, we focused on clarifying the role of m(6)A modification in pituitary ACTH secretion, which is central to stress responses and is also known to be related to depression. Methods: We utilized AtT-20 cell line, mouse-derived pituitary primary culture cells, and patient-derived 3D spheroid cultures of human ACTH-secreting pituitary tumor (ACTHoma) specimens. ACTH concentration in cell supernatants and m(6)A modifications in RNA were quantified using ELISA (MD Bioproducts, and EpigenTek). Pro-opiomelanocortin (Pomc) promoter activity was analyzed using the Dual-Luciferase® Reporter Assay System (Promega). Cell viability was assessed using the RealTime-Glo® MT Cell Viability Assay (Promega). Apoptosis was evaluated by western blotting using Cleaved Caspase-3 expression levels. Results: AtT-20 were incubated with dexamethasone, and the expression of fat mass and obesity-associated (Fto) gene, a m(6)A demethylase, was suppressed, suggesting its involvement in ACTH regulation. Next, Fto knockdown in AtT-20 suppressed Pomc expression by 32% and reduced ACTH concentrations in the supernatant by 14%, with increasing m(6)A modifications. Similarly, treatment with FB23-2, a specific FTO inhibitor that selectively inhibits m(6)A demethylase activity, decreased Pomc expression by 34%, indicating that ACTH production was regulated by m(6)A modification, at least via FTO activity. Next, we investigated the mechanisms by which reduced FTO activity suppressed Pomc expression and found that Pomc promoter activity was reduced and the expression of transcription factors TBX19, Nurr77, Nurr1, and NeuroD1 was suppressed. Surprisingly, when primary cultured mouse pituitary cells were treated with FB23-2, Pomc expression rather increased by 52%, suggesting that the mechanism of ACTH regulation by FTO differs between ACTHomas and normal corticotrophs. When FB23-2 was applied to 3D spheroid cultures of human ACTHoma specimens, the ACTH concentration in the supernatant was suppressed by 15%, supporting this hypothesis. Considering the possibility of FTO as a therapeutic target in ACTHomas, we next performed FB23-2 treatment of 3D spheroid cultures from human ACTHomas, which showed a 14% decrease in cell viability and induction of apoptosis. Discussion: To our knowledge, this is the first study to show that ACTH secretion is mediated by RNA methylation through FTO activity. This regulation differed between normal corticotrophs and ACTHoma cells, suggesting that FTO may be a novel therapeutic target for ACTHomas. Presentation: Sunday, July 13, 2025