Abrogated GADD45b-Mediated Integrity Control of Hematopoietic Stem Cells upon ER Stress and DNA Damage Triggers Lymphopoiesis over Granulopoiesis in Congenital Neutropenia

In patients with severe congenital neutropenia (CN) with maturation arrest of bone marrow granulopoiesis, hematopoietic and myeloid progenitor cells exhibit elevated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) due to inherited mutations in ELANE (neutrophil elastase, NE) or HAX1 (HCLS1-associated protein X-1). UPR and ER stress may induce genetic instability in hematopoietic stem and progenitor cells (HSPCs). Indeed, we recently reported severe changes in cell cycle control and DNA repair pathways (e.g. CHEK1 and BRCA1) in HSPCs of CN patients, as compared to G-CSF treated healthy individuals. We further studied the molecular mechanism of the hyper-sensitivity of CN HSPC to stress conditions. We found that expression of Growth Arrest and DNA-Damage-inducible, beta (GADD45b), which functions as stress sensor regulating cell cycle, survival and apoptosis in response to ER stress or DNA damage, was markedly reduced in myeloid progenitors of CN patients, as compared to G-CSF-treated healthy individuals. GADD45b is essential in stress-induced murine hematopoiesis and myeloid differentiation is severely compromised in gadd45b knockout mice (Gupta S. et al., 2006). Less is known about the functions of GADD45b in the DNA damage response and myeloid differentiation of human HSPCs. To study GADD45b functions in human hematopoiesis, we generated human induced pluripotent stem cells (iPSC) of healthy individuals haplo-insufficient for GADD45b using CRISPR/Cas9-mediated knockout of GADD45b . Human iPSCs were transfected with GADD45b -specific CRISPR/Cas9-gRNA RNP and gene-edited clones were selected for further study. We could not detect any iPSCs clones with frameshift homozygote or compound heterozygote mutations in exon 1 of GADD45b leading to the complete knockout of GADD45b . Most probably GADD45b-deficient iPSCs are lethal. GADD45b^+/- iPSCs also presented a profound phenotype. While these cells retained pluripotency, we detected elevated γH2AX levels in GADD45b^+/- iPSCs upon UV light exposure, as compared to GADD45b-WT cells. In line with up-regulated γH2AX levels, we measured strongly induced DNA damage, as quantified by Lord-Q DNA damage assay. We further analyzed myeloid differentiation of GADD45b^+/- iPSCs using embryoid body (EB)-based method and found that GADD45b^+/- iPSCs cells completely lost the capacity to differentiate into hematopoietic cells. We were able to make EBs of GADD45b^+/- cells, these EBs survived and showed no signs of degradation or cell death, but they produced no floating (hematopoietic) cells. To study the effects of GADD45b inhibition in hematopoietic cells we transfected bone marrow CD34⁺ cells with GADD45b -specific CRISPR/Cas9-gRNA RNP. Similar to iPSCs, we detected markedly elevated UV-induced DNA damage and diminished G-CSF-mediated myeloid differentiation of GADD45b-edited CD34⁺cells in colony-forming units (CFU) assay. These data strongly support the essential role of GADD45b in hematopoietic and myeloid differentiation. Diminished GADD45b expression in CN HSPCs may increase susceptibility to stress and may contribute to malignant transformation.

In order to prevent the appearance and clonal outgrowth of stressed HSPCs with inherited disease-causing mutations in combination with acquired oncogenic mutations, intrinsic DNA damage must be tightly controlled. Failures in the control mechanisms may affect the fate of HSPCs as for instance altered proliferation and wrongly biased differentiation. Therefore, we further evaluated the composition of the HSPC pool in GADD45b-deficient bone marrow cells of CN patients (CN- ELANE n=5, CN- HAX1 n=5), in comparison to healthy individuals treated (n=5) or not (n =7) with G-CSF using multicolor FACS panel of J. Dick. Treatment of healthy individuals with G-CSF induces increase of common myeloid progenitors (CMP, 7-fold) and decrease of multi-lymphoid progenitors (MLP, 10-fold). At the same time, the hematopoietic differentiation in CN patients is substantially shifted from CMP (14-fold decrease) to MLP (36-fold increase) lineage with dramatically reduced numbers of GMPs (3-fold) and increased numbers of B/NK progenitors (5-fold). These data suggest that deregulated integrity control of HSPC pool due to elevated UPR and ER stress and reduced GADD45b expression in hematopoietic cells of CN patients may change composition of HSPC pool and G-CSF response.