ACTHD is defined by an insufficient production of ACTH by the pituitary, and then low adrenal cortisol production. Proper diagnosis and management of ACTHD is crucial as it is a life-threatening condition in the neonatal period characterized by hypoglycemia, cholestatic jaundice, and seizures (Couture et al., 2012). Constitutional ACTHD, i.e., ACTHD diagnosed at birth or during the first years of life, can be isolated or associated with other pituitary hormones deficiencies, such as growth hormone (GH) or thyrotropin-stimulating hormone (TSH), then as part of combined pituitary hormone deficiency (CPHD). ACTHD can be due to mutations in genes coding for transcription factors responsible for pituitary ontogenesis, especially the T-box transcription factor TBX19 (also known as TPIT), NFKB2, LHX3, LHX4, PROP1, HESX1, SOX2, SOX3, OTX2, and FGF8.

Mutations of TBX19 account for approximately two-thirds of neonatal-onset complete isolated ACTHD (Couture et al., 2012). TBX19 is a T-box transcription factor restricted to pituitary pro-opiomelanocortin (POMC)-expressing cells in mice and humans. It is essential for POMC gene transcription and terminal differentiation of POMC-expressing cells (Lamolet et al., 2001). POMC is the precursor of ACTH. Tbx19-deficient mice have only a few pituitary Pomc-expressing cells, with very low ACTH and undetectable corticosterone levels (Pulichino et al., 2003). In humans with isolated ACTH deficiency, TBX19 mutations lead to loss-of-function by different mechanisms, such as the K146R (exon 2, c.437 A>G) pathogenic variant located in the T-box region, which results in a loss of DNA-binding ability (Couture et al., 2012).

Thanks to GENHYPOPIT (Reynaud et al., 2006; Jullien et al., 2019; Jullien et al., 2021), an international network aimed at identifying new genetic etiologies of combined pituitary hormone deficiency, we described the first cases of DAVID syndrome, a rare association of hypopituitarism (mainly ACTHD) and immune deficiency (hypogammaglobulinemia) (Quentien et al., 2012). DAVID syndrome is associated with variants in the nuclear factor kappa-B subunit 2 (NFKB2) gene (Chen et al., 2013; Brue et al., 2014). In particular, we reported that a pathogenic, heterozygous D865G (exon 23, c.2594 A>G) NFKB2 variant was found in a patient presenting with severe recurrent infections from 2 y of age, who at the age of 5 was diagnosed with ACTHD (Quentien et al., 2012). Mutations in the C-terminal region of NFKB2 lead to the disruption of both non-canonical and canonical pathways (Wirasinha et al., 2021; Kuehn et al., 2017; Lee et al., 2014). Full-length NFKB2 (p100) protein has two critical serines, S866 and S870, in the C-terminal domain. Phosphorylation of these sites yields the transcriptionally active NFKB2 (p52) through proteasomal processing of p100 protein. The D865G mutation, located adjacent to the critical S866 phosphorylation site, protects mutant protein from proteasomal degradation, causes defective processing of p100 to p52, and results in reduced translocation of p52 to the nucleus (Chen et al., 2013; Kuehn et al., 2017; Lee et al., 2014). NFKB2^+/D865G resulted in 50% of normal processing to p52, whereas NFKB2^D865G/D865G exhibited near-absence of p52 (Lee et al., 2014). The nuclear factor kappa B (NFKB) signaling pathway is a known key regulator of the immune system (Lee et al., 2014; Lindsley et al., 2014; Carragher et al., 2004), which likely explains the immune phenotype of patients with DAVID syndrome, including susceptibility to infections and auto-immune disorders (Mac et al., 2023). In contrast, the underlying mechanism causing pituitary disorders remains unknown, with two predominating hypotheses: an indirect autoimmune hypophysitis, preferentially affecting corticotroph function, or a primary developmental defect suggested by the expression of NFKB2 in the developing human pituitary (Zhang et al., 2020). However, a developmental role for NFKB2 was not confirmed in the mouse: the Lym1 mouse model carrying a homozygous nonsense variant Y868* presented an apparently normal pituitary development and function (Brue et al., 2014).

Over the last years, human induced pluripotent stem cell (hiPSC)-derived organoids have emerged as promising models to study many developmental mechanisms and their perturbation in disease (Ho et al., 2018). Pioneering work on human embryonic stem cells and later, hiPSC, has established that 3D organoid models can replicate aspects of pituitary development (Ozone et al., 2016; Kasai et al., 2020; Matsumoto et al., 2019), and could thus be of major interest in modeling pituitary disorders (Ozone et al., 2016; Suga et al., 2011; Sasai et al., 2012). In the present study, we applied recent progress in genome editing using CRISPR/Cas9 (Sun and Ding, 2017) to a refined protocol to derive 3D organoids from hiPSC in order to model ACTHD. We validated the recapitulation of corticotroph cell differentiation in vitro by introducing a TBX19 mutation known to induce isolated human ACTHD and documenting the subsequent deficiencies in corticotroph development. When our model was used to characterize the D865G NFKB2 variant found in patients with DAVID syndrome, it displayed dramatically altered corticotroph differentiation in the absence of immune cells, clearly demonstrating for the first time a direct role of NFKB2 in human pituitary development.

After the description of DAVID syndrome as a rare combination of anterior pituitary deficit, mostly ACTHD, with common variable immunodeficiency, we and others found that it is associated with NFKB2 gene mutations affecting specific C-terminal residues of the NFKB2 protein, known to play important roles in immunity and also expressed in the pituitary (Quentien et al., 2012; Chen et al., 2013; Brue et al., 2014). As the mouse model harboring a similar alteration of the ortholog gene lacked an endocrine phenotype (Brue et al., 2014), we wanted to investigate the mechanism of ACTHD in this syndrome. Currently, available in vitro models to study ACTHD and CPHD have strong limits. For instance, studies based on transfections and luciferase-coupled hormone promoters can only give indirect evidence of the effect of a variant of unknown significance as they are based on partly arbitrary amounts of DNA and promoters in an artificial context (Jullien et al., 2019). Murine models only partially replicate human CPHD, as shown for PROP1 mutations and the occurrence of ACTHD (absent in mice but present in 40% of humans) (Castinetti et al., 2016). In the past decade, the reprogramming of differentiated adult cells into induced pluripotent stem cells and advancements in genome editing technologies using CRISPR/Cas9 systems have broadened possibilities for modeling novel aspects of human disease ex vivo (Randolph et al., 2017). Organoid culture has become a powerful tool for modeling otherwise inaccessible congenital human disorders in many organs such as brain (Arnaud et al., 2022), intestine (Schwank et al., 2013), kidney (Takasato et al., 2015), liver (Guan et al., 2017), pancreas (Moreira et al., 2018), ovary (Nanki et al., 2020), and lung (Kong et al., 2021). In the present study, we established a human in vitro model of congenital ACTHD using 3D pituitary organoids differentiated from TBX19 KI and NFKB2 KI hiPSC generated by CRISPR/Cas9 genome editing, in order to compare them with their WT equivalents on the same genetic background. After confirming that our model could replicate pituitary development, we then determined whether it could also replicate a disease, namely ACTHD. As the TBX19^K146R homozygous variant led to ACTHD in humans, we designed a human ‘KI’ organoid model derived from TBX19^K146R/K146R hiPSC. In line with the Tbx19^-/- mouse model (Pulichino et al., 2003), TBX19 KI organoids displayed markedly defective corticotroph differentiation, as compared to WT organoids, thereby validating the methodological approach. Interestingly, in organoids carrying a TBX19^K146R/K146R missense mutation that affects DNA binding, a few corticotrophs were still present, suggesting either a role for TBX19 independent of its DNA binding activity or persistence of residual DNA binding activity with this mutation. This model also raises the possibility for a previously undescribed role of TBX19 during early development and the generation of pituitary progenitors in human. However, as TBX19 mutations have only rarely been associated with other pituitary deficits - especially transient GHD (McEachern et al., 2011), further investigations will be needed.

Using this same approach with NFKB2^D865G/D865G human organoids generated by CRISPR/Cas9 genome edited hiPSC, we demonstrated for the first time a role for NFKB2 in pituitary development. Heterozygous mutations in the C-terminal region of NFKB2, such as D865G, were reported in DAVID syndrome, leading to the disruption of both non-canonical and canonical pathways (Chen et al., 2013; Kuehn et al., 2017; Lee et al., 2014; Lindsley et al., 2014; Liu et al., 2014; Maccari et al., 2017). Even though NFKB2^D865G was identified in a heterozygous state in patients with ACTHD, we decided to use a homozygous model of NFKB2 KI to determine whether NFKB2 was involved in pituitary development and could explain the defect in ACTH production in DAVID syndrome (OMIM#, 615577) (Quentien et al., 2012; Chen et al., 2013; Mac et al., 2023). Indeed, while concomitant common variable immune deficiency (CVID) can be explained by the key roles of NFKB signaling in the immune system, the mechanism of the endocrine deficits caused by NFKB2 mutants was unknown. In mouse models, deletion of Nfkb2 causes abnormal germinal center B-cell formation and differentiation (Carragher et al., 2004). However, the Lym1 mouse model, which carries a truncating variant Y868* in the Nfkb2 orthologue that prevents its cleavage into a transcriptionally active form, had apparently normal corticotroph function and ACTH expression in the pituitary in both heterozygotes and homozygotes (Brue et al., 2014), despite reduced fertility and more severe immune abnormalities in mutant NFKB2 homozygotes (Tucker et al., 2007). Using pituitary organoids differentiated from a NFKB2 KI hiPSC line in comparison with its unmutated control and in the absence of an immune response, we have demonstrated that human corticotroph development is directly disrupted by the missense mutation found in DAVID syndrome. The absence of global alteration of the NFKB signaling pathway shows that mutant p100/p52 protein is directly responsible for the observed phenotype.

The precise molecular mechanisms by which NFKB2 mutation impairs corticotroph development remain to be determined. However, our results (summarized in Figure 14) suggest both early effects on the maturation of pituitary progenitors, on epithelial to mesenchymal transition, and late effects on corticotroph differentiation. First, The NFKB2 gene product p52 could be involved in the early steps of pituitary development. RNA-seq data on day 48 of NFKB2 KI organoid development showed increased HESX1 and BMP4 transcription, in favor of a blockade in an undifferentiated state evocative of the Rathke’s pouch epithelial primordium: indeed, down-regulation of HESX1 is necessary for proper pituitary development (Cohen et al., 2003). This is also suggested by RNA-seq results showing downregulation of PITX1, HES1, LHX3, and FGF10 in mutant organoids, whereas their expression is necessary for progression to a progenitor state (Ohuchi et al., 2000; Ericson et al., 1998). The shift in the BMP4/FGF10 balance favors the idea that the impaired differentiation of pituitary progenitors is at least in part of hypothalamic origin. Our results also suggest a disorganized EMT process, and the need for a functional p100/p52 to mediate the action of EMT inducers on CDH1 downregulation. Finally, concordant with the hypothesis of a differentiation blockade, upregulation of epithelial markers such as CDH1 and KRT8 was observed in mutant NFKB2 KI organoids on day 48. In contrast, RNA-seq showed no change in Wnt and Hedgehog signaling pathways in NFKB2 KI organoids.

Figure 14

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The NFKB2 gene product p52 could also be involved in the final steps of corticotroph differentiation. While qRT-PCR showed increased levels of TBX19 mRNA in mutant organoids, this did not lead to the expected increase in POMC mRNA transcripts. Moreover, some NFKB2 mutants had TBX19 + cells without ACTH expression. This suggests that p52 is necessary for POMC synthesis, which NFKB2^D865G may prevent. This could be due to an abnormal DNA binding of the NFKB heterodimer to the POMC promoter (Giraldi et al., 2011; Drouin, 2016), but as other NFKB binding sites are found in intronic regions, it may also be involved in changes in chromatin structures required for proper transcription. The same possibilities apply to PCSK1, the gene coding for the enzyme necessary for POMC to ACTH conversion, and to other genes involved in corticotroph maturation. Besides, NFKB2^D865G could also block TBX19 + cells in a pre-differentiated state of corticotroph cells expressing FST through its derepression of chromatin remodeling factors such as EZH2 (De Donatis et al., 2016).

GH deficiency has been reported in some patients with heterozygous NFKB2 mutations (Mac et al., 2023). The drastic effect on POU1F1 expression we observed is likely to be the consequence of different phenomena. First, the impaired development of progenitors, and the disruption of PROP1 expression, possibly due to the absence of p52. Second, both FOXO1 and NEUROD4 have p52 binding regions and were differentially expressed. Alongside POU1F1, these two genes have been described as crucial members of a feedforward loop controlling somatotroph function (Stallings et al., 2024). The 12-fold increase in CGA expression and increased expression of pre-gonadotropes or mature thyrotropes (GATA2) and mature gonadotropes (GATA2, CYP11A1, and FOLR1) could be an argument in favor of a redirection of pituitary progenitors towards these lineages. However, the lack of differential expression of NR5A1 contradicts this hypothesis. A more likely explanation is that CGA repression by TBX19 may directly require p52.

The organoid model described in this article presents several advantages over other approaches. It is a true human model of ACTHD, emphasizing the limits of mouse models, as exemplified by the Lym1 model, which showed that Nfkb2 p100/p52 function was not essential for pituitary development in mice, but did not provide insights for humans. Nfkb2^Lym1/Lym1 mice and our NFKB2 KI model have different but functionally very similar mutations, as they both lead to an abnormal processing of p100 with its accumulation leading to a strong reduction of p52 content. The phenotype of Nfkb2^Lym1/Lym1 being more severe than in mice lacking the complete NFKB2 protein (Nfkb2^-/-), these dominant-negative mutations have been called ‘super repressors’ (Tucker et al., 2007). In light of our results, it is surprising that no pituitary deficiencies have been observed in Nfkb2^-/- or Nfkb2^Lym1/Lym1. This suggests that in humans, NFKB2 plays an important role during pituitary development, therefore, constituting a major inter-species difference. Studying p52 binding regions across different species may help understand how this role appeared during evolution.

All patients with DAVID syndrome harbour heterozygous NFKB2 mutations. Whether this reflects embryonic lethality of the mutation at the homozygous state or just the rarity of this genotype is still an unresolved question. However, ‘super repressor’ mutations have been shown to disturb both canonical and non-canonical signalling pathways even when heterozygous (Wirasinha et al., 2021; Kuehn et al., 2017). As our results show impaired POU1F1-dependent lineage development in addition to the severe corticotroph deficit, further experiments on organoids derived from NFKB2^WT/D865G hiPSCs should tell if there are differences in the sensitivity of different lineages to the dominant-negative protein. If so, this could give us clues about why DAVID patients suffer from isolated ACTHD in their vast majority rather than CPHD.

Despite some technical limits, CRISPR/Cas9-based genome editing of hiPSC is a powerful approach to elucidate gene function in congenital pituitary deficiency patients, as was shown for the role of hypothalamic OTX2 regulation of pituitary progenitor cells in congenital pituitary hypoplasia (Matsumoto et al., 2019). While the prevalence of pathogenic variants of known genes in primary hypopituitarism is currently estimated at below 10% (Jullien et al., 2021), exome and whole-genome sequencing regularly identify new genes and variants, for which pathogenicity remains uncertain (Dobin et al., 2013). This model could thus become in the following years the gold standard to confirm the involvement of a given gene in pituitary development, or to classify new variants of unknown significance as likely benign or pathogenic. Finally, pituitary organoids derived from human embryonic stem cells have been transplanted subcutaneously into mice with hypopituitarism; grafted cells were able to secrete ACTH and respond to corticotropin-releasing hormone stimulation (Sasaki et al., 2023), paving the way for innovative substitutive treatments in patients with hypopituitarism (Taga et al., 2023). Conversely, a limitation of this model is the long duration of the differentiation period (approximately 3 months) and the fact that not all hiPSC clones lead to full differentiation of hypothalamic-pituitary organoids despite similar conditions of culture. For these reasons, we could not include confirmation of our results on an independent clone in the present paper.

In conclusion, we have designed and validated a model replicating human constitutive ACTHD using a combination of CRISPR/Cas9 and directed differentiation of hiPSC into 3D hypothalamic-pituitary organoids. Not only has this proof-of-concept reproduced the known prerequisite for the TBX19 transcription factor in human corticotroph differentiation, but it has also allowed a new classification of a NFKB2 variant of previously unknown clinical significance as pathogenic in pituitary development.

From a clinical viewpoint, this organoid model could provide evidence for the pathogenic role of new candidate genes or variants investigated in patients where none other is available, especially in a rare disease affecting a poorly accessible organ like the pituitary gland. From a physiological viewpoint, modeling corticotroph deficiency using hiPSC allows us to find evidence for a functional role for NFKB2 in human pituitary differentiation.

Future studies investigating the direct role of p52 on transcription and changes in chromatin landscape during pituitary development will be necessary to elucidate its precise mechanism of action, as well as the level of pathogenicity of the NFKB2^D865G mutations in its heterozygous form.

RNA-seq results are included as a supporting file (Figure 9—source data 1).

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Author details

1. Thi Thom Mac

   1. Aix-Marseille University, INSERM, UMR1251, Marseille Medical Genetics, Institut MarMaRa, Marseille, France
   2. Hanoi Medical University Hospital, Hanoi, Viet Nam

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3746-2669

2. Teddy Fauquier

   Aix-Marseille University, INSERM, UMR1251, Marseille Medical Genetics, Institut MarMaRa, Marseille, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Methodology, Writing - review and editing

   For correspondence

   teddy.fauquier@univ-amu.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1132-9402

3. Nicolas Jullien

   Aix-Marseille University, CNRS, UMR7051, Institut de NeuroPhysiopathologie, Marseille, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

4. Pauline Romanet

   1. Aix-Marseille University, INSERM, UMR1251, Marseille Medical Genetics, Institut MarMaRa, Marseille, France
   2. Aix-Marseille University, APHM, INSERM, MMG, Laboratory of Molecular Biology, La Conception Hospital, Institut MarMaRa, Marseille, France

   Contribution

   Validation

   Competing interests

   No competing interests declared

5. Heather Etchevers

   Aix-Marseille University, INSERM, UMR1251, Marseille Medical Genetics, Institut MarMaRa, Marseille, France

   Contribution

   Validation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0201-3799

6. Anne Barlier

   1. Aix-Marseille University, INSERM, UMR1251, Marseille Medical Genetics, Institut MarMaRa, Marseille, France
   2. Aix-Marseille University, APHM, INSERM, MMG, Laboratory of Molecular Biology, La Conception Hospital, Institut MarMaRa, Marseille, France
   3. Aix Marseille University, APHM, INSERM, MMG, Department of Endocrinology, La Conception Hospital, Institut MarMaRa, Marseille, France

   Contribution

   Supervision, Validation

   Competing interests

   No competing interests declared

7. Frederic Castinetti

   1. Aix-Marseille University, INSERM, UMR1251, Marseille Medical Genetics, Institut MarMaRa, Marseille, France
   2. Aix Marseille University, APHM, INSERM, MMG, Department of Endocrinology, La Conception Hospital, Institut MarMaRa, Marseille, France

   Contribution

   Conceptualization, Supervision, Validation

   Competing interests

   No competing interests declared

8. Thierry Brue

   1. Aix-Marseille University, INSERM, UMR1251, Marseille Medical Genetics, Institut MarMaRa, Marseille, France
   2. Aix Marseille University, APHM, INSERM, MMG, Department of Endocrinology, La Conception Hospital, Institut MarMaRa, Marseille, France

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Project administration, Writing - review and editing

   For correspondence

   Thierry.BRUE@ap-hm.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8482-6691

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