Loss-of-Function Mutation in tet2 in Zebrafish Leads to Early MDS like Phenotype

Abstract Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia. Loss-of-function mutations in the Ten-11 Translocation 2 (TET2) gene are implicated in MDS and AML, but evidence that TET2 mutations are also found in healthy individuals as a result of clonal hematopoiesis calls into question the contribution of TET2 to leukemogenesis. Many studies have tried to mutate the DNA binding domain in TET2 both in mouse and zebrafish, but the blood phenotypes observed have been inconsistent. We created a tet2 zebrafish mutant using CRISPR-Cas9 technology, where we deleted 2.1 kb from exon 2, confirmed by cDNA analysis. A recent publication suggests the importance of Ser99 in the stability of the TET2 protein (Wu et al., 2018) and this conserved serine residue is contained in the region of deletion in our zebrafish tet2 mutant. These tet2 mutant fish were incrossed to create a maternal-zygotic mutant with no tet2 expression. Importantly, quantitative PCR demonstrated that there was no compensatory effect from tet1 and tet3 in the context of tet2 loss. Whole mount in situ hybridization performed on 24-48h tet2 mutant zebrafish embryos provided evidence of a significant reduction of early and mature myeloid and erythroid cells through staining for spi1/pu.1 (myeloid), gata1 (erythroid) myeloperoxidase (mpx; neutrophils), carboxypeptidase A5 (cpa5; mast cells) and l-plastin (lcp1; macrophages). The stem cell compartment was not impacted, as shown by wild-type runx1/c-myb expression. Further, o-dianisidine staining revealed low hemoglobin content in tet2 mutants compared with control embryos. Overall, the early embryos elucidate a refractory anemia like phenotype. Flow cytometry analysis of kidney marrow from 3-month-old adult tet2 mutant fish similarly demonstrated a decrease in the myeloid and erythroid compartments, in keeping with characteristics typical of low stage MDS. We are currently employing RNAseq and MeDIP analysis on blood cell populations from tet2 mutants to determine the genetic and epigenetic signatures associated with this mutation. This zebrafish model of tet2 loss uniquely provides with a dynamic phenotype that starts with refractory anemia with progression through low stage MDS. Thus this model will be beneficial in elucidating the mechanisms underlying how tet2 contributes to MDS and AML and provide a preclinical platform to screen for therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

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