Early Clonal Hematopoiesis Detected in Gata2 heterozygosity By Color-Barcoding of Hematopoietic Stem Cells in Zebrafish

Germline GATA2 haploinsufficiency underlies an inherited syndrome of predisposition to myeloid malignancies. The mechanism by which GATA2 mutations lead to leukemia in children and young adults remains to be determined. We generated gata2b heterozygous mutant zebrafish using CRISPR/Cas9 technology to study this process. We used a color-barcoding system in zebrafish called Zebrabow that labels each hematopoietic stem cell (HSC) born during embryogenesis with a different color. Color-barcoding was induced by a blood-specific promoter-driven Cre recombinase during development at 24 hours post fertilization when there are on average 20 HSC clones. We then examined adult hematopoiesis in wildtype and gata2b^+/- zebrafish. Surprisingly, at steady state the marrow of gata2b^+/- fish showed color dominance with a single color clone contributing to over 30% of granulocytes, while the myeloid output in wildtype clutchmates was polyclonal. This suggested a baseline oligoclonal state in gata2b^+/- hematopoiesis. Gata2b^+/- fish also had a concomitant mild myelocytopenia with 20% reduction of myeloid cells in the marrow as early as 2 months post fertilization (mpf), reminescent of monocytopenia often present in GATA2 patients. Myelocytopenia with concurrent expansion of progenitor population was more profound in gata2b^-/- fish with up to 4-fold increase of progenitor to myeloid cell ratio at 3mpf. Gata2b^-/- fish are adult viable, possibly due to partial compensation by a duplicated gata2a gene. Germline GATA2-associated MDS and AML often harbor additional acquired mutations in epigenetic regulators. To study the effect of somatic mutations in GATA2 heterozygosity, we induced mosaic mutagenesis in wild-type or gata2b^+/-embryos by injecting 1-cell embryos with Cas9 mRNA and guide RNAs targeting zebrafish orthologs of ASXL1 and STAG2, both of which have been associated with GATA2 mutations in MDS or AML. The marrow analysis of injected gata2b^+/- fish showed an increase of hematopoietic progenitor cells and worse myelocytopenia at 7 mpf compared to control injected wildtype fish (progenitor/myeloid ratio of 1.7, p < 0.05). Single color expansion was observed in over 60% of injected gata2b^+/-fish as measured by Shannon index of diversity, and correlated with stag2a and stag2b frame-shift mutations with variant allele fraction of >20%. This mutations result in the predicted loss of at least 2 of the 4 total stag2 alleles. In summary, gata2b deficiency alone results in aberrant hematopoiesis with reduced HSC diversity and myelocytopenia. Mutations in stag2 lead to early enhanced clonal dominance in gata2b^+/- but not wildtype fish. Our studies suggest a model in which germline GATA2 mutations lead to a state of reduced HSC diversity during development. Such a state predisposes to clonal events due to acquired mutations in epigenetic regulators resulting in clonal hematopoiesis and myeloid leukemias, establishing a link between abnormal developmental hematopoiesis and leukemia risk later in life.