Page 161 - Haematologica March 2020

Modeling chronic myeloid leukemia in zebrafish
all patients with CML.7,8 This fusion protein is a constitu- tively active tyrosine kinase that persistently activates var- ious signaling pathways regulating cell proliferation, trans- formation, and survival, thereby promoting leukemogene- sis.9 Further research and exploration are needed to recog- nize the blast crisis of CML since the specific mechanism leading to it is not yet fully understood.
The therapeutic use of tyrosine kinase inhibitors (TKI), such as imatinib, dasatinib, and bosutinib, has trans- formed the management of CML, largely turning a lethal disorder into a chronic condition. However, conventional TKI therapy for CML still presents challenges, including the appearance of TKI-resistant BCR/ABL1 mutants10 and the relative resistance of CML leukemia stem cells (LSC)11 to TKI. In addition, all TKI have a similar spectrum of toxic effects4 that can negatively affect the patient’s quali- ty of life. Furthermore, CML and other malignancies include a population of cancer stem cells (CSC) that is able to regenerate or self-renew, resulting in therapeutic resist- ance and disease progression, and the inability to eradicate these CSC remains a significant obstacle to curing these diseases.
Biomedical research requires suitable animal disease models in which to study the mechanisms responsible for the cellular and molecular pathologies, and for testing cer- tain therapeutic methods. There are high levels of conser- vation in terms of genomics, histoembryology, physiolo- gy, cardiac electrophysiology, and drug metabolic path- ways between zebrafish and humans,12 and zebrafish thus represent a possible model for studying hematopoietic development and for high-throughput drug screening. However, there is currently no zebrafish CML model. The construction of a zebrafish CML model would expand our ability to study this disease and to develop new drugs that could benefit CML patients.
Methods
Zebrafish husbandry
All experiments involving zebrafish were carried out in accor- dance with the guidelines set by the Institutional Animal Care and Use Committee of Southern Medical University, Guangzhou, China. Zebrafish were raised, bred, and staged according to stan- dard protocols.13,14 The following strains were used: AB (wild-type strain, WT) and Tg(lyz:DsRed).15
GenerationofthepToLhsp70:p210BCR/ABL1 construct and of Tg(hsp70:p210BCR/ABL1) transgenic zebrafish
The transgenic construct consisted of the zebrafish heat shock protein (Hsp) 70 promoter, human BCR/ABL1 (hBCR/ABL1) (b3a2) cDNA, Tol2 elements, and the SV40 polyA sequence. We cloned hsp70 promoter elements by polymerase chain reaction (PCR) using hsp70-specific primers 5′-GTATCGATTCAGGGGT- GTCGCTTGGT-3′ and 5′-CCGATATCACCGGTCT- GCAGGAAAAAAAAAC-3′. The hBCR/ABL1 (b3a2) cDNA frag- ment was isolated from the plasmid NGFR P21016 (Addgene) after digestion with EcoRI. The hsp70 promoter sequence was then placed upstream of the hBCR/ABL1 (b3a2) cDNA and subcloned into the pToL vector with minimal Tol2 elements and an SV40 polyA sequence to form the pToL hsp70:p210BCR/ABL1 construct. The transgenic line was generated by injecting 50 pg of the pToL hsp70:p210BCR/ABL1 construct together with Tol2 transposase mRNA into zebrafish embryos at the one-cell stage. Founders were iden- tified by PCR confirmation of the transgene.
Western blot
Protein was extracted from whole embryos at 6 days post-fer- tilization (dpf) or from blood cells from the kidney marrow (KM) of 1-year-old Tg(hsp70:p210BCR/ABL1) and age-matched WT controls. Proteins were quantified, and assessed by western blot analysis. Protein lysates were probed with rabbit anti-c-Abl antibody (1:1000 dilution, Cell Signaling Technology). Mouse anti-glycer- aldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:5000 dilution, Cell Signaling Technology) was included as an internal control.
Cell proliferation assay
Wild-type (WT) and Tg(hsp70:p210BCR/ABL1) embryos at 3 dpf and 1-year old adults were incubated in 10 mM BrdU (Sigma-Aldrich) for 2 hours (h) and 4 h, respectively. The embryos and KM blood cells were stained with mouse-anti-BrdU antibody (Roche) and rabbit-anti-Lcp1 antibody (gift from Dr. Zilong Wen),17 followed by Alexa Fluor 555-anti-mouse antibody (Invitrogen) and Alexa Fluor 488-anti-rabbit antibody (Invitrogen) for fluorescent visuali- zation.
Terminal deoxynucleotidyl transferase dUTP nick end labeling assay
Transferase dUTP nick end labeling (TUNEL) assay was carried out using an In Situ Cell Death Detection Kit (TMR red, Roche), followed by rabbit anti-Lcp1 antibody and Alexa Fluor 488-anti- rabbit antibody (Invitrogen) for fluorescent visualization.
Transplantation
Whole KM cell suspensions were prepared from Tg(lyz:DsRed) and Tg(hsp70:p210BCR/ABL1-lyz:DsRed) (CML-like) fish. Three days after receiving a sublethal dose of radiation (25 Gy), 0.2 million cells were injected intracardially into irradiated WT recipients using a glass capillary needle (World Precision Instruments).
Drug treatment
Embryos were soaked in egg water containing 1‰ dimethylsul- foxide (DMSO) (Sigma-Aldrich), 20 mmol/L imatinib (Selleck), 5 mmol/L dasatinib (Selleck), 10 mmol/L bosutinib (Selleck), 20 mmol/L LY364947 (MedChemExpress), 2.5 mmol/L FTY720 (MedChemExpress), 0.5 mmol/L BEZ235 (MedChemExpress), or compounds from a compound library (TargetMol) for drug treat- ment.
Statistical analysis
Data were analyzed using SPSS software (version 20). Differences between two groups were analyzed using Student t-tests and differences among multiple groups by one-way analy- sis of variance (ANOVA) with Tukey’s adjustment. Significance was accepted when P<0.05. Data were expressed as mean±Standard Error of Mean (SEM).
Details of other methods used are available in the Online Supplementary Appendix.
Results
Transient expression of humanized BCR/ABL1 increased the number of myeloid cells in zebrafish larvae
The BCR/ABL1 fusion gene is present in nearly all cases of CML. Protein sequence comparisons revealed that zebrafish Bcr and Abl1 shared around 71% and 73% iden- tities, respectively, with their human counterparts and contained a highly conserved kinase domain on Abl1
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