SAT-014 ATAC sequencing analysis elucidates novel GHRH-Induced DNA Damage pathways in Murine Somatotrophs

Abstract Disclosure: D. Watts: None. S. Song: None. F.M. Ruf-Zamojski: None. S. Melmed: None. A. Ben-Shlomo: None. Background: Although excess DNA damage is abundant in growth hormone (GH) secreting pituitary adenomas, underlying mechanisms remain unresolved. We previously showed that long-acting GH-releasing hormone (GHRH) agonist CJC-1295 (CJC), stimulates GH secretion via cAMP-induced DNA damage in murine somatotrophs. We now aimed to elucidate pathways subserving CJC -induced somatotroph DNA damage. Methods: Adult male C57BL/6 mice were injected subcutaneously with 10 μg/kg CJC or PBS (n=20 each) thrice weekly for 8 weeks in two independent experiments. Following treatments, dissected pituitaries were snap frozen, and 3 pituitaries per condition were pooled for single nucleus assays. For the first cohort, we performed single nuclei ATAC-Seq (snATACSeq) only. Results were validated in the second cohort using single nucleus multiome. In both groups, nuclei were isolated at the Cedars-Sinai Genomics Core and processed for 10X Genomics assays followed by sequencing. Differentially accessible genes and their associated pathways were identified through computational analysis, and selected results validated by western blotting. Results: CJC treatment increased plasma IGF-1 levels in all mice as expected. SnATACSeq clustering identified 12 cell type clusters per experiment, with somatotrophs identified based on Gh, Ghrhr, and Pou1f1 markers. Motif enrichment analysis of CJC-treated somatotrophs revealed 197 and 175 enriched motifs in each cohort, with 106 common motifs, including transcription factors (TFs) linked to GH production Ep300 (p=0.00088), AP-1 (p=0.00056), and Pou1f1 (6.01E-10). Additional enriched motifs were associated with the FOXO pathway which included Forkhead Box proteins (FOXO1 p=0.030, FOXO3 p=0.019, FOXO4 p= 0.00036) and retinoic acid receptors RAR-α p = 4.03*10-29, RAR-β p=5.15*10-11, RXR-a p=2.58*10-9). The KEGG pathway analysis showed a significant change in the FOXO signaling pathway, p=0.000017). GO pathway analysis of the enriched motifs identified 1045 overlapping pathways across the two experiments. Response to retinoic acid (p=2.626*10-10) and retinoic acid receptor signaling pathway (1.67*10-9, and nuclear retinoic acid receptor binding (p=3.4*10-9) were key significantly changed pathways. Western blot analysis confirmed increased RAR-α and decreased pFOXO1. Summary: -Increased enriched motifs for TFs were identified in somatotrophs of mice treated with CJC. Prolonged treatment with a long-acting GHRH agonist increases GH production and enhances somatotroph chromatin accessibility to FOXO and retinoic acid TFs. As both these pathways are implicated in DNA damage and repair, their signaling may underlie mechanisms for GHRH-induced DNA damage in murine somatotrophs. Presentation: Saturday, July 12, 2025