Modeling GATA2 Deficiency in Mice: The R396Q Mutation Disrupts Normal Hematopoiesis

GATA2 deficiency is a rare, autosomal dominant germline disorder of immune dysfunction with a high likelihood for bone marrow failure and leukemic transformation early in life, as it accounts for up to 7% of pediatric myelodysplastic syndrome (MDS) and 15% of advanced MDS cases. Several sequencing studies have identified common cytogenetic abnormalities and secondary somatic mutations thought to contribute to leukemic progression, such as monosomy 7, trisomy 8, and mutations in ASXL1, SETBP1, or STAG. However, the mechanisms of disease progression have been difficult to identify due to a lack of disease-specific experimental models. Using CRISPR/Cas9 gene editing, we generated a murine model of one of the most common GATA2 mutations associated with leukemic progression in GATA2 deficiency, Gata2^R396Q/+, and assessed the effects of this mutation on hematopoietic function and development.

Mutant mice exhibited a modest decrease in white blood cell counts throughout life, driven mostly by reductions in lymphocytes, neutrophils, and monocytes, with no differences in red blood cell or platelet counts. Examination of the bone marrow compartment of adult mice revealed changes in the composition of hematopoietic progenitors, including a significant reduction in the absolute numbers of all committed myeloid and lymphoid progenitors. Among the hematopoietic stem and progenitor cells (HSPC), there were intrinsic biases toward the long-term hematopoietic stem cell (HSC) and multipotent progenitor (MPP) 2 populations at the expense of the short-term HSC population, driven mostly by a significant increase in CD150 expression. To determine the functional potential of Gata2^R396Q/+ bone marrow HSCs, we performed bone marrow transplants. While repopulating ability was not completely ablated, Gata2^R396Q/+ bone marrow exhibited a significant reduction in engraftment after primary and secondary transplantation, with a slight bias toward myeloid production in secondary transplants.

To gain a better understanding of Gata2^R396Q/+ HSPCs, we performed single-cell RNA sequencing on bone marrow Lineage^-Sca1⁺c-Kit⁺ (LSK) cells. In support of our functional studies, Gata2^R396Q/+ LSK cells showed a significant reduction in hscScore, and Gene Set Enrichment Analysis (GSEA) revealed a loss of stemness and an accelerated aging phenotype compared to Gata2^WT LSKs. Gata2^R396Q/+ LSK cells also exhibited a myeloid bias. Coupled with our GSEA analysis, transplantation studies, and significant increase in CD150 expression on HSPCs, these data suggest that the R396Q mutation induces an aged HSC compartment.

Given the importance of GATA2 in the generation of definitive HSCs and the predominance of GATA2 deficiency in pediatric/adolescent leukemia, we also assessed the effects of Gata2^R396Q/+ on early hematopoiesis. Colony forming assays and transplantation experiments revealed significant defects in HPSC function in the fetal liver of E12.5 and E14.5 Gata2^R396Q/+ embryos. Importantly, the intrinsic biases and increased CD150 expression seen in bone marrow HSPCs were apparent in fetal liver HSPCs by E14.5, suggesting that the hematopoietic defects seen in adult mice had a developmental origin. Imaging of the E10-10.5 aorta gonad mesonephros (AGM) revealed a significant reduction of intra-aortic hematopoietic cluster formation in Gata2^R396Q/+ embryos. Likewise, Gata2^R396Q/+ E11-E11.5 AGMs were unable to repopulate irradiated recipients after transplantation, suggesting a loss of definitive HSC production. Indeed, limiting dilution transplants from Gata2^R396Q/+ adult bone marrow showed a three-fold reduction in HSC number, indicating that the R396Q mutation reduces the pool of lifelong HSCs.

Together, our data show that the R396Q mutation reduces the pool of definitive HSCs and induces an accelerated aging phenotype, directing HSCs toward a more myeloid-biased progenitor. Both phenotypes could be potential contributors toward leukemic transformation in patients, and our model provides a useful tool to study the mechanisms of malignant transformation in GATA2 deficiency.

Crispino:Cellarity: Consultancy; Alethiomics: Membership on an entity's Board of Directors or advisory committees; Syndax: Research Funding.