Modeling corticotroph deficiency with pituitary organoids supports the functional role ofNFKB2in human pituitary differentiation

Abstract Background Deficient Anterior pituitary with common Variable Immune Deficiency (DAVID) syndrome is a rare condition characterized by the association of adrenocorticotropic hormone deficiency (ACTHD) and primary hypogammaglobulinemia, caused by NFKB2 heterozygous mutations. Nuclear factor kappa B (NFKB) signaling is a key regulator of the immune system; however, the underlying mechanism of its association with endocrine symptoms remains unknown. Two main hypotheses explain the effects of mutant NFKB2 on the pituitary gland: an autoimmune hypophysitis, preferentially affecting corticotroph function, or a primary developmental defect. The role of NFKB2 in the development of the human pituitary was called into question by Nfkb2 -deficient Lym1 mice, which have normal pituitary functions. Purpose The aim of this study was to create a human disease model to define the role of NFKB2 in human pituitary development. Methods We established pituitary organoids in three dimensions (3D) culture after directed differentiation from CRISPR/Cas9-edited human induced pluripotent stem cells (hiPSC). First, we conducted a proof-of-concept study, introducing a homozygous TBX19 K 146 R/K 146 R missense pathogenic variant in hiPSC, an allele found in patients with congenital isolated ACTHD. Then, we used the same method to produce NFKB2 D 865 G/D 865 G mutant organoids, harboring the pathogenic missense variant previously identified in DAVID patients. This mutation causes a failure of NFKB2 p100 phosphorylation that blocks processing to form active NFKB2 p52. We then characterized pituitary organoid development by transcriptomics using bulk RNA sequencing and quantitative RT-PCR, and by immunofluorescence in section and whole-mount. Results Analysis of wild-type (WT) organoids demonstrated that this in vitro model recapitulates corticotroph cell differentiation. TBX19 K 146 R/K 146 R organoids conserved early expression of HESX1 , but had significantly decreased PITX1 , TBX19 , LHX3, and POMC transcription . NFKB2 D 865 G/D 865 G organoids also had dramatically reduced corticotrophs. Furthermore, NFKB2 D 865 G/D 865 G perturbs the normal expression of 66 genes known to contribute to pituitary development, among which 21 transcription factors. Conclusions We used a combination of CRISPR/Cas9 editing and refinement of a 3D organoid culture protocol to model human ACTHD due to TBX19 or NFKB2 mutations. The NFKB2 variant studied induced a significant decrease in corticotroph differentiation, demonstrating for the first time a direct functional role of NFKB2 in human pituitary development. Signaling through NFKB2 is thus a valid new candidate pathway in the pathogenesis of isolated or syndromic ACTHD.