Page 163 - Haematologica March 2020

Modeling chronic myeloid leukemia in zebrafish
increased after overexpression of hBCR/ABL1 compared with the control group. Expression levels of markers of more mature neutrophils, such as lyz, mpx, and SB, also increased significantly. Patients with CML typically devel- op a highly characteristic differential white blood cell (WBC) count with high concentrations of myelocytes and segmented neutrophils. The current results implied that hBCR/ABL1 expression in zebrafish may promote myelo- cytes and may be capable of inducing myeloid leukemia in vivo.
hBCR/ABL1 was inherited in Tg(hsp70:p210BCR/ABL1) transgenic zebrafish
Tg(hsp70:p210BCR/ABL1) transgenic zebrafish were created using a construct (Figure 2A) expressing hBCR/ABL1 under the control of the zebrafish heat shock-inducible hsp70 promoter.18,19 The construct was designed to integrate the complete coding sequence into the host genome using the Tol2 transposition system, allowing the generation of multiple lines of transgenic zebrafish. Tg(hsp70:p210BCR/ABL1) transgenic zebrafish founders were confirmed by PCR (Figure 2B). Stable F1 Tg(hsp70:p210BCR/ABL1) transgenic zebrafish were obtained by intercrossing founder fish and were confirmed by sequencing (data not shown). F2 and the offsprings were obtained by mating F1 fish with WT fish. The tem- porospatial expression of hBCR/ABL1 in Tg(hsp70:p210BCR/ABL1) transgenic zebrafish was evaluated by WISH (Figure 2C). hBCR/ABL1 expression was appar- ent throughout the body of Tg(hsp70:p210BCR/ABL1) embryos at 3 dpf after heat shock treatment. Further detection by real-time-quantitative (RT-q)-PCR showed that levels of hBCR/ABL1 mRNA were significantly elevated after heat shock treatment in both coro1a:GFP+ blood cells from Tg(hsp70:p210BCR/ABL1) transgenic zebrafish embryos and hematopoietic progenitors and myelocytes in KM blood cells from Tg(hsp70:p210BCR/ABL1) transgenic zebrafish adults (Online Supplementary Figure S1). The hBCR/ABL1 oncogene encoding the p210BCR/ABL1 protein was also detected in Tg(hsp70:p210BCR/ABL1) transgenic zebrafish embryos and adult kidneys (Figure 2D). The molecular weight of p210BCR/ABL1 measured in vitro (Online Supplementary Figure S2) confirmed that the weight of the fusion protein generated was as expected. The p210BCR/ABL1 protein was highly expressed in Tg(hsp70:p210BCR/ABL1) transgenic zebrafish after heat-shock treatment.
Inducible hBCR/ABL1 expression in Tg(hsp70:p210BCR/ABL1) transgenic zebrafish promoted myeloid lineage in zebrafish embryos
Expression of p210BCR/ABL1 induces leukemia and myelo- proliferative disorders, indicating a direct, causal role of BCR/ABL in CML.9,20-23 We established Tg(hsp70:p210BCR/ABL1) transgenic zebrafish with expression of p210BCR/ABL1 and stable inheritance. To further explore the function of p210BCR/ABL1 in zebrafish, we observed its influence on hematopoietic development in zebrafish embryos using WISH and cytochemical staining with lin- eage-specific markers (Figure 2E). The numbers of lcp1+ pan-myeloid cells, lyz+ neutrophils, SB+ neutrophils, and mfap4+ macrophages were significantly increased in Tg(hsp70:p210BCR/ABL1) transgenic zebrafish larvae at 3 dpf compared with WT controls. This suggested that hBCR/ABL1 expressed in zebrafish could either promote the production of HSC or their differentiation into each
hematopoietic lineage. To distinguish between these pos- sibilities, we detected the HSC marker (cmyb), erythro- cyte marker (βe1), and lymphocyte marker (rag1). There was no difference in the number of cmyb+ HSCs between Tg(hsp70:p210BCR/ABL1) transgenic zebrafish and WT con- trols at 36 hpf, but the number was significantly increased in transgenic zebrafish at 60 hpf (Online Supplementary Figure S3A-D, I and J). Numbers of βe1+ erythrocytes and rag1+ lymphocytes were significantly decreased in the transgenic zebrafish compared with the WT control zebrafish at 5 dpf (Online Supplementary Figure S3E-H). These findings suggest that hBCR/ABL1 may promote myeloid differentiation.
Inducible hBCR/ABL1 expression in Tg(hsp70:p210BCR/ABL1) adult zebrafish created phenotype resembling human CML
The natural progression of untreated CML is bi- or triphasic, with the initial CP followed by AP, BP, or both. CP is characterized by leukocytosis in both the PB and BM, and a preponderance of granulocytes in various degrees of maturation. However, blasts account for <2% of the peripheral WBC and <5% of the nucleated cells in the BM.24 As the disease progresses, patients enter the AP followed by the BP, during which there is hematopoietic differentiation arrest, allowing immature blasts to accu- mulate in the BM and spill into the circulation. A level of 10-19% of blasts in the PB or BM marks the transition from CP to AP, along with a predominance of promyelo- cytes. A level of at least 20% PB or BM blasts indicates the progression to the BP.24 To explore the possibility of developing leukemia-like hematologic disorders in Tg(hsp70:p210BCR/ABL1) adult fish, PB and KM cells were col- lected from Tg(hsp70:p210BCR/ABL1) and WT fish at 6 months to 1-year old and subjected to cytological and WBC analyses (Table 1 and Figure 3A and B). Seventy-seven of 101 (76.24%) Tg(hsp70:p210BCR/ABL1) adult zebrafish devel- oped CML-like disease, including 68 with a CML-CP phe- notype, marked by massive leukocytosis in the PB or KM, including increased percentages of myelocytes and myeloid precursors. In the early stage of CML-CP, the increased leukocytes were primarily neutrophils in vari- ous degrees of maturation. Myelocytes accounted for >15% in the PB or >50% in the KM, with blasts usually accounting for <2% of the PB and <5% of the KM during this phase. We referred to this period as CML-CP1 (Table 2). Differentiation was then interrupted in the late stage of CML-CP as the condition progressed towards CML- AP. The increased leukocytes were primarily myeloid pre- cursors and blasts, with myeloid precursors >10% and blasts >2% in the PB, and myeloid precursors >15% and blasts >5% in the KM. We referred to this period as CML- CP2 (Table 2). Eight of the 77 CML-like Tg(hsp70:p210BCR/ABL1) transgenic zebrafish showed CML- AP phenotype including significant 2- to 10-fold increases in the percentages of blasts and myeloid precursors, with blasts >10% in the PB or KM. Amongst the 77 CML-like Tg(hsp70:p210BCR/ABL1) adult zebrafish, one progressed to CML-BP with >90% blasts expanding in both the PB and KM. We also identified some phenotypes accompanying these CML-like Tg(hsp70:p210BCR/ABL1) adult zebrafish, including eosinophilia, lymphocytosis and thrombocyto- sis (Figure 3C). Six of 77 (7.79%) CML-like Tg(hsp70:p210BCR/ABL1) adult zebrafish presented with eosinophilia, with eosinophils accounting for >0.1% of
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