FLT3ITD and IDH2 mutations synergistically promote myeloid skewing and differentiation arrest in a zebrafish model of acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) is a heterogeneous group of diseases with highly diverse mutational profiles. Animal models to examine the cooperative effects of mutation combinations in AML with high throughput are much needed. The conserved molecular regulation of hematopoiesis between zebrafish and human has made zebrafish uniquely suitable for this purpose. In this study, we generated transgenic zebrafish carrying both FLT3ITD (Fms-Like Tyrosine Kinase 3-Internal Tandem Duplication) and Isocitrate Dehydrogenase 2 mutations IDH2R140Q or IDH2R172K, expressed under Runx1 promoter and incorporated into genome via Tol2 transposons system. Transgenic embryos co-expressing FLT3ITD and IDH2 mutations showed significant increase in markers associated with HSC (cmyb, runx1) and neutrophils (l-plastin, mpo and SBB staining). As these embryos were raised to adulthood, around 50% of them developed AML with increased number of Mpo+ (myeloperoxidase) blasts in the kidney marrow (KM) and peripheral blood (PB) at 6-12 months of age. Transplantation of KM cells from the leukemic fish into irradiated wildtype recipient fish resulted in aggressive leukemic phenotype in the recipients and shortened survival. Single-cell transcriptome of the leukemic fish and their wildtype siblings showed that genes associated with cell proliferation, metabolism and immune responses, were significantly upregulated. Myeloid skewing began at the level of hematopoietic stem cell (HSC) and became more apparent at multipotent progenitor (MPP). On the other hand, differentiation of myeloid and erythroid progenitors were perturbed. These AML models were clinically relevant, as the leukemic phenotypes in both embryos and adults could be ameliorated by quizartinib (FLT3 inhibitor) and enasidenib (IDH2 inhibitor). In conclusion, transgenic zebrafish carrying FLT3 and IDH2 mutations recapitulated the morphologic, clinical, transcriptomic and functional characteristics of human AML, providing high throughout and cost effective models for rapid drug discovery for AML.