Establishment of a novel zebrafish platform for the study of mutation co-operation in acute myeloid leukaemia (AML)

Abstract

Acute myeloid leukaemia (AML) is a group of heterogeneous diseases with distinct clinicopathologic and genetic features. The unique genetic mutational profile in individual AML patients may affect its response to treatment and hence clinical outcome. Remarkably, recurrent patterns of co-mutations supported the proposition of cooperativity in driving leukaemogenesis. This project aims to develop a novel zebrafish platform that allows rapid evaluation of the impact of different genetic mutation combinations on leukemogenesis and their drug response. Zebrafish embryos were co-injected with a dual combination of mutations that are prevalent in human AML viz. FLT3-ITD with IDH2 R140Q, IDH2 R172K, IDH1 R132H, DNMT3A R882H and NPM1 Type A mutation at 25 pg per embryos for each gene, and their effects on primitive myelopoiesis were evaluated. Singe expression of these mutant genes at the same dose showed no effects on myelopoiesis. Co-expression of FLT3-ITD and IDH2 R140Q in zebrafish embryos uniquely induced expansion of myeloid progenitors, as evidenced by an increase in pu.1+ population-based on whole-mount in-situ hybridisation and increase in Sudan Black B staining based on cytochemistry. Co-expression of FLT3-ITD with DNMT3A R882H, IDH1 R132H, IDH2 R172K or NPM1 Type A mutation showed no synergistic effect. Transgenic fish lines, in which FLT3-ITD/IDH2 R140Q, FLT3-ITD/IDH2 R172K and FLT3-ITD/IDH1 R132H were expressed under Runx1 enhancer regulation, were generated by Tol2 transgenesis. At six months of age, Tg(Runx1: FLT3ITDIDH2R140Q) fish, but not Tg(Runx1: FLT3ITDIDH1R132H) or Tg(Runx1: FLT3ITDIDH2R172K) fish, showed an increase in myeloid precursors compared with wildtype or Tg(Runx1: FLT3ITD) fish. Mechanistically, co-expression of FLT3-ITD and IDH2 R140Q showed no potentiation of downstream FLT3 signalling or hydroxyglutarate (2HG) levels. As a proof-of-principle, zebrafish embryos co-injected with FLT3-ITD and IDH2 R140Q were used to screen a list of FDA approved and preclinical drugs for their effects on myelopoiesis. The effects of these drugs on wildtype embryos were also compared. Drugs that reduced myelopoiesis in “dual expressors” but not wildtype embryos include Enasidenib and Dasatinib. Interestingly, the effects of FLT3 inhibitor Quizartinib were attenuated when IDH2 R140Q was co-expressed with FLT3-ITD. In conclusion, this project has demonstrated zebrafish as a powerful model for studying co-operative mutations in AML and predict drug response, paving the way to personalised treatment.