Mice Expressing a Germline Gata2T354M Mutation Phenocopy the GATA2 Deficiency Syndrome of Humans

GATA2 Deficiency Syndrome is caused by germline loss-of-function mutations in the transcription factor GATA2. Clinical manifestations include reduced monocytes and other immune cell populations, opportunistic infections, and/or the development of myeloid malignancies. GATA2 mutations are heterozygous in ~98% of these patients (PMID: 30190467), and the remaining WT allele is epigenetically silenced in nearly all leukemias that arise in these individuals (PMID: 33831168). Three broad classes of mutations lead to GATA2 deficiency syndrome: missense mutations in the zinc finger domains, frameshift or nonsense mutations throughout the protein coding region, or partial deletions of the +9.5 kb intronic enhancer. Among the missense mutations, GATA2^T354M confers the strongest predisposition to develop acute myeloid leukemia (AML) or myelodysplastic syndromes (83% of cases) (PMID: 28642594). In mice, deficiency or reduction of GATA2 expression leads to reduced hematopoietic stem cell numbers and function (PMIDs: 8078582, 22996659). In addition, we have demonstrated that Gata2 acts as a tumor suppressor in mice, since the somatic inactivation of Gata2 cooperates with PML::RARA to induce a highly penetrant form of acute promyelocytic leukemia with a median leukemia-free survival of 120 days (PMID: 34125173). However, it remains unclear how germline GATA2 mutations increase the risk of myeloid malignancies.

To develop a pre-clinical model of GATA2 Deficiency Syndrome, we generated germline Gata2^T354M knock-in mice, derived from two independent founders. Hematopoietic precursor cells from Gata2^T354M/+ heterozygous mice express the Gata2^T354M and Gata2^WT alleles at equal levels, defined by single-cell RNA sequencing (scRNA-seq). Flow cytometry revealed that the bone marrow of Gata2^T354M/+ mice shows a 35.2% reduction in monocytes (p<0.0001 by t-test) and a 39.5% reduction in long-term hematopoietic stem cells (LT-HSCs; p=0.033 by t-test), compared to WT littermates. In the periphery, no detectable changes were observed in the major hematopoietic cell populations. However, donor bone marrow cells from Gata2^T354M/+ mice showed a severe disadvantage in competitive bone marrow transplantation experiments. One month following transplantation of Gata2^T354M/+ and WT bone marrow cells at an input ratio of 1:1, we observed a 5.7:1 ratio of WT to Gata2^T354M/+ cells in HSCs, monocytes, B cells, T cells, NK cells, and dendritic cells (p<0.0001 by t-test). This suggests that Gata2^T354M/+ impairs the ability of HSCs to home and/or engraft in recipient mice. To determine the transcriptional changes induced by Gata2^T354M, we performed scRNA-seq analysis of lineage-depleted bone marrow from 57-week-old mice, and identified 155 differentially expressed genes (53 upregulated and 102 downregulated; fold-change ³ 2 and FDR < 0.05 by ANOVA) between Gata2^T354M/+ and WT granulocyte monocyte progenitors (GMPs). GMPs from Gata2^T354M/+ mice also showed coordinate changes in the expression of 288 genes that have previously been identified as dysregulated in hematopoietic precursors from GATA2 deficiency syndrome patients, including Mpo, Elane, and Myb (FDR=0.038 by permutation test) (PMID: 32556286). To date, we have also aged 20 unmanipulated Gata2^T354M/+ mice beyond one year; one died unobserved at 21 months, and had a massively enlarged spleen (1.8 g), suggestive of either a spontaneous leukemia or a myeloproliferative process. In sum, germline Gata2^T354M/+ knock-in mice recapitulate many of the phenotypes observed with GATA2 Deficiency Syndrome in humans, including defects in hematopoietic stem/progenitors and monocytes, and many of the same transcriptional changes. Remarkably, we have recently learned that these mice can be bred to homozygosity. Gata2^T354M/T354M mice show a 1.7-fold decrease in the frequency of B cells in the blood compared to WT and Gata2^T354M/+ mice, thereby suggesting that homozygosity may cause a more severe phenotype than heterozygosity. Gata2^T354M mice therefore represent an important new model to study the impact of germline GATA2 mutations that are known to alter hematopoiesis, and predispose patients to develop myeloid malignancies. The complete characterization of the preleukemic phenotypes of both heterozygous and homozygous mice is well underway, and studies to define key factors associated with leukemic progression are in progress.