Steiner, J. P. | bioRxiv Science

bioRxiv2025

Phosphoproteomic dysregulation drives tumor proliferation in Cushing's disease

Pituitary adenomas constitute up to 20% of primary brain tumors, yet somatic mutations are only found in 15% of pituitary adenomas. Epigenomic dysregulation has been proposed as a tumorigenic mechanism in pituitary adenomas causing Cushings disease (CD). We created paired datasets of human CD adenomas and en-route margin adult human pituitary glands and assayed their chromatin accessibility, DNA methylation, transcriptomic, proteomic and phospho-proteomic landscapes. In CD adenomas, we found epigenetic reactivation of a neurodevelopmental phosphoprotein program typically lost in the post-natal pituitary gland. CD cells overexpressed PPP1R17, a potent endogenous inhibitor of the ubiquitous protein phosphatase PP2A. Mechanistically, PPP1R17 overexpression in normal murine pituitary cells recapitulated the adenoma phenotype, and PPP1R17-mediated tumorigenesis was reversible using an FDA-approved small molecule PP2A agonist both in-vitro and in-vivo. Our findings highlight aberrant peptide phosphorylation as a targetable mechanism in CD. Significance statementCushings disease (CD) causes significant morbidity and mortality despite best medical and surgical treatment. Surgery is the mainstay of treatment, but carries perioperative risks and is frequently followed by remission. There is a paucity of effective medical treatments, due in part to a limited understanding of tumor mechanisms. The majority of CD adenomas are wild-type, with no known causal mutations. Our study identifies phosphoproteomic dysregulation as a mechanism of CD tumorigenesis common to wild-type and mutant CD adenomas. We target this pathway in-vivo and in-vitro using an FDA-approved small molecule. Our study proposes a novel therapeutic strategy for patients with CD.

cancer biology↗

bioRxiv2025

3D organoids containing endothelial and neural cells generation by serial inductions of differentiation on human iPSC-derived embryoid bodies

3D brain organoids have been widely used as a tool to study human brain development and disorders. Although endothelial cells play important roles in the brain development and pathogenesis in neurological disorders, most 3D brain organoids lack inherent endothelial cells and need either the addition of endothelial cells or to be transplanted to animals to reconstitute such vascular structures, likely missing the developmental interactions of endothelial cells and other cells in the human brain. In order to reconstitute a 3D organoid mimicking the in vivo neural and endothelial cells development, we cultured iPSC-derived embryoid bodies in sequentially applied endothelial and neuronal induction media along with Matrigel embedding. The resulting 3D organoid consists of both neural cells and endothelial cells with vascular like structures, as determined by immunostaining. With scRNA-Seq analysis, the brain organoid was confirmed to contain neural cell types similar with human brains, including a variety of excitatory and inhibitory neurons and glia. Furthermore, when compared with traditional cerebral organoids without endothelial cells using RNA-Seq analysis, the endothelial containing neural organoids (EC-neural organoids) showed difference in gene profiles and favored angiogenesis and vasculogenesis. Of the differentially expressed genes, KRBA2 expression was found higher in neural cells and its inhibition by siRNA treatment resulted in decreased transcriptions of a variety of genes such as neuronal differentiation specific genes but not in genes specific to pluripotent stem cells such as OCT4. The EC-neural organoids also express receptors to SARS-CoV-2 similar to human brains. This 3D model provides a useful tool to study the interactions of endothelial cells and neural cells in the brain development and neural infectious disorders where endothelial cells and pericytes play pivotal roles.

neuroscience↗