Page 167 - Haematologica March 2020

Modeling chronic myeloid leukemia in zebrafish
Figure S4). After incubation with these TKI for 48 h, we calculated the numbers of lcp1+ myeloid cells in WT and Tg(hsp70:p210BCR/ABL1) larvae in the posterior blood island (PBI) region at 5 dpf (Figure 6A and B). All the TKI signif- icantly reduced the number of lcp1+ myeloid cells in Tg(hsp70:p210BCR/ABL1) larvae compared with the DMSO control group. In addition, lower concentrations (20 and 40 mmol/L) of imatinib significantly reduced the expand- ed lcp1+ myeloid population in Tg(hsp70:p210BCR/ABL1) lar- vae, but the number of lcp1+ myeloid cells was also signif- icantly reduced in WT larvae at higher concentrations (80 mmol/L) compared with DMSO (Online Supplementary Figure S5). These results suggest that high doses of ima- tinib may affect normal myelopoiesis, which may be associated with more adverse events or unpredictable off- target effects. Further studies are needed to clarify these effects and to support the clinical treatment of patients with CML.
We screened a library of 171 compounds in 3 dpf WT and Tg(hsp70:p210BCR/ABL1) embryos to examine their abili- ty to reverse the disease phenotype. We reduced the incu- bation time to 24 h to speed up the screening process, and then calculated the numbers of lcp1+ myeloid cells in WT and Tg(hsp70:p210BCR/ABL1) larvae in the PBI region at 4 dpf. Ten inhibitors, including the natural compound, icaritin, as well as CC-223, BEZ235, AZD3759, icotinib, DB07268, NQDI-1, selonsertib (GS-4997), LY364947 and ciliobrevin A (HPI-4) effectively reduced the expanded lcp1+ myeloid population in Tg(hsp70:p210BCR/ABL1) embryos compared with DMSO-treated controls (Figure 6C).
Discussion
We constructed a new germline of transgenic zebrafish expressing the hBCR/ABL1 fusion protein. Expression of hBCR/ABL1 in Tg(hsp70:p210BCR/ABL1) transgenic zebrafish altered hematopoiesis by up-regulating myeloid genes, as detected in larvae at 3 dpf. Adult Tg(hsp70:p210BCR/ABL1) transgenic zebrafish developed CML characterized by clonal myelocytic blasts, representing the first zebrafish model of hBCR/ABL1-induced CML. As the disease pro-
gressed, hematopoietic differentiation was interrupted, and immature blasts and myeloid precursors accumulated in the BM and spilled into the circulation in this zebrafish model, closely resembling the natural course of human CML progression without treatment. The most accurate CML animal model to date is the SCLtTA/BCR-ABL mouse line21 established by Koschmieder et al. in 2005. However, these mice only survive for 4-17 weeks, while adult Tg(hsp70:p210BCR/ABL1) transgenic zebrafish could sur- vive for from 12 to >18 months, with or without heat shock, which was longer than all previous mouse models. The incidence of CML in the Tg(hsp70:p210BCR/ABL1) trans- genic model was increased by hBCR/ABL1 heat shock. This Tg(hsp70:p210BCR/ABL1) transgenic model may thus provide insights into the mechanism that drives the tran- sition from CML-CP to CML-AP or CML-BP.
Tyrosine kinase inhibitors (imatinib, dasatinib, and bosutinib) effectively reduced the expanded myeloid population in Tg(hsp70:p210BCR/ABL1) embryos, suggesting that the pharmacological pathways in this model were similar to those in human CML. The discovery of ima- tinib has greatly improved the longevity and quality of life of patients with CML; however, some patients devel- op resistance to TKI and may even progress toward CML- AP or CML-BP of the disease despite TKI therapy. Second- and third-generation TKI were developed to treat patients in whom imatinib fails, with up to 40-87% of patients achieving durable complete cytogenetic remis- sion.4 However, more serious side-effects have recently been associated with these second- and third-generation TKI. Understanding the underlying cause of resistance and screening for novel targeted drugs with low toxicity and high efficiency thus remain important steps in com- bating CML. Further studies are planned to generate site- directed mutations of the ABL1 kinase domain in Tg(hsp70:p210BCR/ABL1) transgenic zebrafish using gene-edit- ing technology (such as CRISPR/Cas9). The ABL1 kinase domain is frequently mutated in clinical cases, and exam- ination of these mutants may thus help to elucidate the mechanism responsible for TKI resistance.
In the present study, we screened a compound library and discovered 10 new targeted drugs that reduced the
Table 2. Hemogram and classification of Tg(hsp70:p210BCR/ABL1) fish in chronic myeloid leukemia chronic phase. Classification Group Number Percentages in PB (%) Percentages in KM (%)
Location
KM
PB KM and PB KM
PB
Leukemia cell types
Neutrophils in various degrees of maturation
Myeloid precursors
Blasts Myeloid Myelocytes Lymphocytes Blasts Myeloid Myelocytes Lymphocytes
Normal 55
CML-CP1 I 27
0.06 ± 0.04
0.00 ± 0.00
0.15 ± 0.15 0.38 ± 0.22 0.78 ± 0.31
2.45 ± 1.31‡
precursors
1.68 ± 0.39
1.29 ± 0.42
3.27 ± 1.53 3.48 ± 1.52 3.40 ± 0.86
12.22 ± 3.83†
8.09 ± 1.45
4.09 ± 1.16
20.11 ± 7.08* 17.79 ± 2.38* 3.03 ± 0.67
8.26 ± 5.07
90.17 ± 1.55
94.62 ± 1.36
76.46 ± 8.51 78.36 ± 2.63 92.79 ± 1.26
77.07 ± 7.69
4.41 ± 0.56
1.42 ± 0.42
2.57 ± 0.93 2.79 ± 0.83 8.35 ± 1.24‡
0.71 ± 0.57
precursors
12.56 ± 0.76
8.15 ± 0.49
10.68 ± 2.16 12.82 ± 2.38 19.07 ± 1.47†
11.47 ± 4.02
46.89 ± 1.29
58.75 ± 0.90*
43.93 ± 3.51 52.06 ± 2.34* 37.61 ± 2.36
39.07 ± 3.72
36.14 ± 1.21
31.68 ± 1.04
42.82 ± 3.86 32.32 ± 1.59 34.98 ± 2.17
48.75 ± 8.09
CML-CP 2
II 4 III 19 IV 15
V 3
White blood cell counts were obtained by identifying at least 500 cells per kidney marrow (KM) preparation and at least 1,500 cells per peripheral blood (PB) preparation.The percentages were indicated by mean±Standard Error of Mean.Chronic myeloid leukemia-chronic phase (CML-CP) 1:blasts <2%,myeloid precursors <10% in PB or blasts <5%,myeloid precursors <15% in KM.CML-CP 2:blasts >2%,myeloid precursors >10% in PB or blasts >5%, myeloid precursors >15% in KM. CML-CP 1-Group I: myelocytes increased in KM. CML-CP 1-Group II: myelocytes and myeloid precursors increased in PB. CML-CP 1-Group III:myelocytes increased in both PB and KM,myeloid precursors increased in PB.CML-CP 2-Group IV:myeloid precursors and blasts increased in KM.CML-CP 2-Group V:myeloid precursors and blasts increased in PB. *Indicates myelocytes in PB or KM increased by >15% or 50%.†Indicates myeloid precursors in PB or KM by >10% or >15%.‡Indicates blasts in PB or KM increased by >2% or >5%.
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