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

Abstract

AbstractBackgroundDeficient 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 byNFKB2heterozygous 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 byNfkb2-deficientLym1mice, which have normal pituitary functions.PurposeThe aim of this study was to create a human disease model to define the role ofNFKB2in human pituitary development.MethodsWe 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 homozygousTBX19K146R/K146Rmissense pathogenic variant in hiPSC, an allele found in patients with congenital isolated ACTHD. Then, we used the same method to produceNFKB2D865G/D865Gmutant 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.ResultsAnalysis of wild-type (WT) organoids demonstrated that thisin vitromodel recapitulates corticotroph cell differentiation.TBX19K146R/K146Rorganoids conserved early expression ofHESX1, but had significantly decreasedPITX1,TBX19,LHX3,andPOMCtranscription. NFKB2D865G/D865Gorganoids also had dramatically reduced corticotrophs. Furthermore,NFKB2D865G/D865Gperturbs the normal expression of 66 genes known to contribute to pituitary development, among which 21 transcription factors.ConclusionsWe used a combination of CRISPR/Cas9 editing and refinement of a 3D organoid culture protocol to model human ACTHD due toTBX19orNFKB2mutations. TheNFKB2variant 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.