GADD45β plays an essential role in the G-CSF triggered granulocytic differentiation of human hematopoietic stem cells

Abstract

Congenital neutropenia (CN) is a bone marrow failure syndrome caused by inherited gene mutations in e.g., ELANE or HAX1 leading to markedly low neutrophil numbers in peripheral blood. The mechanism of the “maturation arrest” of myeloid progenitors in CN patients is not fully elucidated. We aimed to shed light on the pathomechanism of this bone marrow failure. Considering the fact that inherited mutations may disturb the fitness of hematopoietic stem and progenitor cells (HSPCs) and may cause deregulated differentiation, we first investigated the CN HSPC composition and stress levels. Indeed, we found that early HSCs showed higher stress levels than later progenitors. Moreover, despite G-CSF treatment, CN HSPCs were committed towards lymphopoiesis, rather than myelopoiesis. We identified the myeloid differentiation primary response (MyD) and stress sensor gene GADD45β that might play a role in the defective stress-induced granulopoiesis: G-CSF induced the expression of GADD45β in healthy individuals, but not in CN patients. We functionally elaborated the role of GADD45β in stress response and granulopoiesis by CRISPR/Cas9-mediated knockout in iPSCs, primary HSPCs, and zebrafish. Since CRISPR/Cas9 gene-editing in iPSCs and HSPCs is challenging, we established a method that allows the fluorescent labeling of CRISPR/Cas9 RNP and thus the enrichment of gene-edited cells. GADD45β knockout cells were significantly more susceptible to UV-mediated DNA damage compared to control cells. Knockout of GADD45β leads to drastic reduction of granulocytes in vitro and in vivo. At the same time, the ectopic expression of GADD45β in CN HSPCs restored granulocytic differentiation. We could show that GADD45β is responsible for the demethylation and thus induction of genes essential for granulocytic differentiation. Among others, GADD45β activates the retinoic acid signaling pathway to induce granulopoiesis. Strikingly, the treatment of CN HSPCs with ATRA could bypass the GADD45β activation and rescued diminished neutrophil differentiation. Taken together, we could show that GADD45β regulates granulopoiesis downstream of G-CSF by modulating retinoic acid signaling. To further elaborate the mechanisms of defective granulopoiesis in CN, we established an iPSC model. Research on rare diseases requires valuable patient samples, which are very restricted in the case of pediatric patients. iPSC models may overcome these limitations. This model allows us to study CN pathogenesis, but also to test novel therapies. Using the iPSC model, we were able to set up a CRISPR/Cas9-based ELANE knockout to restore granulocytic differentiation. This approach could serve as gene therapy for CN patients.