Functional study of zebrafish Flt3 in embryonic hematopoiesis with particular reference to modeling human FLT3/ITD AML

Abstract

FMS-like tyrosine kinase 3 (FLT3) is expressed in human hematopoietic stem and progenitor cells (HSPC) but its role during embryogenesis is unclear. In acute myeloid leukemia (AML), internal tandem duplications (ITD) of FLT3 at the juxtamembrane domain (JMD) (FLT3/ITD) occur in 30% of patients and are associated with inferior clinical prognosis. Tyrosine kinase domain (TKD) mutations (FLT3/TKD) occur in 5% of cases and their prognostic significance is unclear. Both mutations result in constitutive activation of FLT3 signaling. In this project, zebrafish was used to examine the role of flt3 in developmental hematopoiesis and model human FLT3/ITD+ and FLT3/TKD+ AML in this organism. The JMD and TKD regions of zebrafish Flt3 were remarkably similar to their mammalian counterparts. Knockdown of flt3 by morpholino significantly reduced the expression of l-plastin (pan-leukocyte), csf1r and mpeg1 (macrophage) as well as that of c-myb (definitive HSPC), lck and rag1 (T-lymphocyte) shown by whole mount in situ hybridization (WISH). Expression of human FLT3/ITD in zebrafish embryos by plasmid DNA injection resulted in Stat5, Erk1/2, and Akt phosphorylation and the expansion of myeloid cells (pu.1+, mpo+, and cebpα+). The latter was ameliorated by FLT3 inhibitor quizartinib (formerly AC220). Human FLT3/TKD (D835Y) induced significant, albeit modest, myeloid expansion resistant to quizartinib, corroborating with their clinical responses to FLT3 inhibitors.

Intriguingly, injection of FLT3/ITD mRNA induced dorsal axis duplication with duplicated expression of notochord marker col9a2 at 24 hours-post-fertilization (hpf). The effects could be ameliorated by quizartinib. To identify the initiating signals associated with axis duplication, expression of genes associated with dorsoventral patterning was examined at shield stage (6 hpf). The Spemann’s organizer was expanded as evidenced by increased and ectopic expression of goosecoid (gsc), chordin (chd) and follistatin (fst). The up-regulation of Fst was confirmed by quantitative RT-QPCR and Western Blot. Interestingly, FST protein expression and STAT5 phosphorylation were significantly up-regulated in FLT3/ITD-transfected Hela cells, as well as in BaF3 cells expressing FLT3/ITD and FLT3/ITD+ AML cell lines MV4-11 and MOLM-13. FST transcript, protein expression (in cell) and protein secretion (in serum) were significantly increased in FLT3/ITD+ AML samples. Importantly, quizartinib significantly inhibited FST expression and FST knockdown significantly reduced leukemia growth of MV4-11 cells in vitro.

This study provided novel insight to the role of flt3 during hematopoiesis, established a zebrafish model expressing of FLT3/ITD and FLT3/TKD mutation from AML, and identified FST as a novel molecular target of FLT3/ITD+ AML whose therapeutic potential should be further evaluated.