OR20-05 Generation Of CDH23-mutated Pituitary Neuroendocrine Tumor Organoids From Induced Pluripotent Stem Cells To Model Cushing’s Disease Pathogenesis

Disclosure: J. Chakrabarti: None. S. Mallick: None. J. Churko: None. J. Eschbacher: None. A.S. Little: None. K.C. Yuen: None. Y. Zavros: None. Background: Cushing's disease (CD) is a debilitating disease caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor (PitNET). While most corticotropic PitNETs occur sporadically, rarely CD arises in the context of genetic familial syndromes. CDH23 syndrome is clinically associated with the development of Usher syndrome. In a study that linked mutations in CDH23 with familial and sporadic PitNETs suggested that these genetic alterations could play a role in the pathogenesis of CD. Patient-derived induced pluripotent stem cells (iPSCs) resemble germline mutations of affected individuals, can be differentiated into several tissues, and are used to study hereditary diseases. Our published studies (Mallick et al., 2023, Translational Research) demonstrated that organoids generated from a CD patient expressing CDH23 mutation (iPSC(CDH23) organoids) exhibited tumor behavior consistent with the expected pathology and function of corticotroph PitNET from patients. Objective: To generate iPSCs from CD patients expressing CDH23 (iPSC(CDH23)) to reveal the disease pathogenesis under directed differentiation to PitNET organoids. Methods: Single cell RNA sequencing (scRNA-seq) was used to compare the transcriptomic profile of iPSC(CDH23), iPSC organoids generated from a healthy individual (hPit(iPSC)), and patient PitNET tissue generated organoids (hPitNET41(org) and hPitNET42(org)). Lineage trajectory analysis was used on scRNA-seq data collected at 15, 30 and 60 days during the iPSC differentiation program. Results: Markers of dysregulated cell cycle and increased proliferation were identified in iPSC(CDH23), hPitNET41(org) and hPitNET42(org) organoids. The expression of the E2 factor (E2F) family of transcription factors were upregulated in the PitNET organoids compared to hPit(iPSC). Noteworthy, was an increase in the CD44/xCT(SLCA7A11) antioxidant system expression in the iPSC(CDH23), hPitNET41(org) and hPitNET42(org) cultures. Using lineage trajectory analysis using iPSC(CDH23) and hPit(iPSC) cultures, a base cluster enriched in Wnt/NOTCH+ stem cell markers was selected to direct the lineage trajectories for the other clusters. Trajectories for hPit(iPSC) cultures revealed multiple branching paths, leading to clusters enriched for markers of pituitary cell lineages (lactotroph, somatotroph, thyrotroph, gonadotroph and corticotroph). The trajectories for iPSC(CDH23) organoids exhibited two predominant paths from the base cluster to terminal clusters that were highly enriched with markers representative of corticotroph and NOTCH signaling. Conclusion: PitNET organoids generated from iPSCs of CD patients reveal a dysregulation in the cell lineage differentiation pathways leading to a skewed development of corticotrophs. Understanding the molecular mechanisms of the pathogenesis of CD will identify therapeutic targets for patients. Presentation: Saturday, June 17, 2023