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Human BCR/ABL1 induces chronic myeloid leukemia-like disease in zebrafish

Chronic myeloid leukemia (CML) is induced by the BCR/ABL1 oncogene, which encodes a protein tyrosine kinase. We examined the effect of direct overexpression of the human p210BCR/ABL1 oncoprotein in zebrafish. Humanized p210BCR/ABL1 protein was detectable in Tg(hsp70: p210BCR/ABL1) transgenic zebrafish embryos and adult kidney marrow. Transgenic zebrafish developed CML, which could be induced via cells transplanted into recipients. The expression of human BCR/ABL1 promoted myeloid lineages in Tg(hsp70:p210BCR/ABL1) transgenic embryos. A total of 77 of 101 (76.24%) Tg(hsp70:p210BCR/ABL1) adult transgenic zebrafish (age 6 months-1 year) developed CML. CML in zebrafish showed a triphasic phenotype, similar to that in humans, involving a chronic phase predominantly characterized by neutrophils in various degrees of maturation, an accelerated phase with an increase in blasts and immature myeloid elements, and a blast phase with >90% blasts in both the peripheral blood and kidney marrow. Tyrosine kinase inhibitors, as the standard drug treatment for human CML, effectively reduced the expanded myeloid population in Tg(hsp70:p210BCR/ABL1) transgenic embryos. Moreover, we screened a library of 171 compounds and identified ten new drugs against BCR/ABL1 kinase-dependent or -independent pathways that could also reduce lcp1+ myeloid cell numbers in Tg(hsp70:p210BCR/ABL1) transgenic embryos. In summary, we generated the first humanized zebrafish CML model that recapitulates many characteristics of human CML. This novel in vivo model will help to elucidate the mechanisms of CML disease progression and allow high-throughput drug screening of possible treatments for this disease.

Ferrata Storti Foundation C hronic myeloid leukemia (CML) is induced by the BCR/ABL1 oncogene, which encodes a protein tyrosine kinase. We examined the effect of direct overexpression of the human p210 BCR/ABL1 oncoprotein in zebrafish. Humanized p210 BCR/ABL1 protein was detectable in Tg(hsp70: p210 BCR/ABL1 ) transgenic zebrafish embryos and adult kidney marrow. Transgenic zebrafish developed CML, which could be induced via cells transplanted into recipients. The expression of human BCR/ABL1 promoted myeloid lineages in Tg(hsp70:p210 BCR/ABL1 ) transgenic embryos. A total of 77 of 101 (76.24%) Tg(hsp70:p210 BCR/ABL1 ) adult transgenic zebrafish (age 6 months-1 year) developed CML. CML in zebrafish showed a triphasic phenotype, similar to that in humans, involving a chronic phase predominantly characterized by neutrophils in various degrees of maturation, an accelerated phase with an increase in blasts and immature myeloid elements, and a blast phase with >90% blasts in both the peripheral blood and kidney marrow. Tyrosine kinase inhibitors, as the standard drug treatment for human CML, effectively reduced the expanded myeloid population in Tg(hsp70:p210 BCR/ABL1 ) transgenic embryos. Moreover, we screened a library of 171 compounds and identified ten new drugs against BCR/ABL1 kinasedependent or -independent pathways that could also reduce lcp1 + myeloid cell numbers in Tg(hsp70:p210 BCR/ABL1 ) transgenic embryos. In summary, we generated the first humanized zebrafish CML model that recapitulates many characteristics of human CML. This novel in vivo model will help to elucidate the mechanisms of CML disease progression and allow highthroughput drug screening of possible treatments for this disease.

Human BCR/ABL1 induces chronic myeloid leukemia-like disease in zebrafish Introduction
Chronic myeloid leukemia (CML) is a malignant bone marrow proliferative tumor originating from hematopoietic stem cells (HSC), with an annual incidence of 1-2/100,000 and accounting for 15-20% of all adult leukemias. 1 CML is characterized by uncontrolled proliferation of myeloid cells and their progenitors in the peripheral blood (PB) and bone marrow (BM). 2 The development of CML progresses from a chronic phase (CP) to an accelerated phase (AP), and finally to a blast phase (BP). Most patients in the CML-CP are clinically asymptomatic, but are diagnosed with leukocytosis characterized by mature granulocytes in the PB and BM. Disease progression to AP and BP is accompanied by a severe reduction in cellular differentiation, with immature blasts displacing mature cells. 3 The final transformation phase can result in both lymphoblastic (25%) and myeloblastic (50%) subtypes, with a further 25% manifesting biphenotypic or undifferentiated phenotypes. 4 The presence of the Philadelphia chromosome (Ph + ) is an important diagnostic indicator for CML. 5 It is generated by a reciprocal translocation between chromosomes 9 and 22, referred to as t(9;22)(q34;q11). 6 This translocation results in the BCR/ABL1 fusion gene, which is translated to the p210 BCR/ABL1 oncoprotein in almost all patients with CML. 7,8 This fusion protein is a constitutively active tyrosine kinase that persistently activates various signaling pathways regulating cell proliferation, transformation, and survival, thereby promoting leukemogenesis. 9 Further research and exploration are needed to recognize 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 transformed 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 mutants 10 and the relative resistance of CML leukemia stem cells (LSC) 11 to TKI. In addition, all TKI have a similar spectrum of toxic effects 4 that can negatively affect the patient's quality 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 resistance 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 certain therapeutic methods. There are high levels of conservation in terms of genomics, histoembryology, physiology, cardiac electrophysiology, and drug metabolic pathways 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.

Zebrafish husbandry
All experiments involving zebrafish were carried out in accordance 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 standard protocols. 13,14 The following strains were used: AB (wild-type strain, WT) and Tg(lyz:DsRed). 15 Generation of the pToL hsp70:p210 BCR/ABL1 construct and of Tg(hsp70:p210 BCR/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 fragment was isolated from the plasmid NGFR P210 16 (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:p210 BCR/ABL1 construct. The transgenic line was generated by injecting 50 pg of the pToL hsp70:p210 BCR/ABL1 construct together with Tol2 transposase mRNA into zebrafish embryos at the one-cell stage. Founders were identified by PCR confirmation of the transgene.

Western blot
Protein was extracted from whole embryos at 6 days post-fertilization (dpf) or from blood cells from the kidney marrow (KM) of 1-year-old Tg(hsp70:p210 BCR/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-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:5000 dilution, Cell Signaling Technology) was included as an internal control.

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-antirabbit antibody (Invitrogen) for fluorescent visualization.

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 analysis 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.

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% identities, respectively, with their human counterparts and contained a highly conserved kinase domain on Abl1 (Ensembl GRCh37 release 92). We evaluated the function of the hBCR/ABL1 oncoprotein in zebrafish by overexpressing hBCR/ABL1 mRNA encoding the p210 BCR/ABL1 oncoprotein. We then detected the numbers of myeloid cells during embryonic hematopoietic development by lcp1, lyz, mpx whole-mount in situ hybridization (WISH), and Sudan Black B (SB) cytochemical staining ( Figure 1). lcp1, also named l-plastin, is a pan-myeloid marker that identifies all myeloid subsets, including macrophages and neutrophils. Numbers of lcp1 + cells were significantly 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 develop 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 myelocytes and may be capable of inducing myeloid leukemia in vivo.

hBCR/ABL1 was inherited in Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish
Tg(hsp70:p210 BCR/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:p210 BCR/ABL1 ) transgenic zebrafish founders were confirmed by PCR ( Figure 2B). Stable F1 Tg(hsp70:p210 BCR/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 temporospatial expression of hBCR/ABL1 in Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish was evaluated by WISH ( Figure 2C). hBCR/ABL1 expression was apparent throughout the body of Tg(hsp70:p210 BCR/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:p210 BCR/ABL1 ) transgenic zebrafish embryos and hematopoietic progenitors and myelocytes in KM blood cells from Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish adults (Online Supplementary Figure S1). The hBCR/ABL1 oncogene encoding the p210 BCR/ABL1 protein was also detected in Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish embryos and adult kidneys ( Figure 2D). The molecular weight of p210 BCR/ABL1 measured in vitro (Online Supplementary Figure S2) confirmed that the weight of the fusion protein generated was as expected. The p210 BCR/ABL1 protein was highly expressed in Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish after heat-shock treatment.

Inducible hBCR/ABL1 expression in Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish promoted myeloid lineage in zebrafish embryos
Expression of p210 BCR/ABL1 induces leukemia and myeloproliferative disorders, indicating a direct, causal role of BCR/ABL in CML. 9,20-23 We established Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish with expression of p210 BCR/ABL1 and stable inheritance. To further explore the function of p210 BCR/ABL1 in zebrafish, we observed its influence on hematopoietic development in zebrafish embryos using WISH and cytochemical staining with lineage-specific markers ( Figure 2E). The numbers of lcp1 + pan-myeloid cells, lyz + neutrophils, SB + neutrophils, and mfap4 + macrophages were significantly increased in Tg(hsp70:p210 BCR/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 possibilities, we detected the HSC marker (cmyb), erythrocyte marker (βe1), and lymphocyte marker (rag1). There was no difference in the number of cmyb + HSCs between Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish and WT controls 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:p210 BCR/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 accumulate 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 promyelocytes. 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:p210 BCR/ABL1 ) adult fish, PB and KM cells were collected from Tg(hsp70:p210 BCR/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:p210 BCR/ABL1 ) adult zebrafish developed CML-like disease, including 68 with a CML-CP phenotype, 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 various 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 precursors 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:p210 BCR/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:p210 BCR/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:p210 BCR/ABL1 ) adult zebrafish, including eosinophilia, lymphocytosis and thrombocytosis ( Figure 3C). Six of 77 (7.79%) CML-like Tg(hsp70:p210 BCR/ABL1 ) adult zebrafish presented with eosinophilia, with eosinophils accounting for >0.1% of  Table S1). This was similar to the "Ph-positive eosinophilic/basophilic CML" described by Goh et al. 24 Figure 3D). In addition, the morbidity of CML-like disease in heat-shock-treated Tg(hsp70:p210 BCR/ABL1 ) adult zebrafish was higher than in non-induced Tg(hsp70:p210 BCR/ABL1 ) adult zebrafish. The ratios of individuals in CML-CP2 and CML-AP were increased among heat-shock-treated Tg(hsp70:p210 BCR/ABL1 ) compared with untreated Tg(hsp70:p210 BCR/ABL1 ) adult zebrafish ( Figure  3E). This result suggests that overexpression of BCR/ABL1 is an important factor in accelerating the course of CML.

Tg(hsp70:p210 BCR/ABL1 ) transgenic fish displayed abnormal myeloid cell expansion resulting from increased proliferation and inhibition of apoptosis
The above results indicated that myeloid cells accumulated in Tg(hsp70:p210 BCR/ABL1 ) fish from the embryonic stage to the adult, which could be caused by accelerated proliferation or reduced apoptosis. To clarify the cellular mechanisms responsible for myeloid cell expansion in Tg(hsp70:p210 BCR/ABL1 ) fish, we monitored myeloid cell proliferation and death by BrdU incorporation and TUNEL assay, respectively. BrdU incorporation was significantly increased in Tg(hsp70:p210 BCR/ABL1 ) larvae and adult KM compared with WT controls, indicating that myeloid cell expansion in Tg(hsp70:p210 BCR/ABL1 ) fish was the result of increased proliferation ( Figure 4A, B, E and F). However, myeloid cell apoptosis was also significantly decreased in Tg(hsp70:p210 BCR/ABL1 ) larvae and adult KM compared with WT controls, suggesting that the expansion of myeloid cells Tg(hsp70:p210 BCR/ABL1 ) was also caused by reduced apoptosis (Figure 4C, D, G and H).

Tg(hsp70:p210 BCR/ABL1 ) transgenic cells with induced CML-like disease were transplantable
To determine the aggressiveness of the leukemia induced by Tg(hsp70:p210 BCR/ABL1 ) activity, whole KM blood cells from Tg(hsp70:p210 BCR/ABL1 ) fish were transplanted into γ-irradiated WT adult hosts and the resulting fish were tested to determine if the CML-like phenotype developed in the Tg(hsp70:p210 BCR/ABL1 ) fish could be transplanted into the WT fish. We used 1-year old Tg(hsp70:p210 BCR/ABL1 -lyz:DsRed) CML-like donors and Tg(lyz:DsRed) control donors, in which the granulocytes were marked by red fluorescence. Each irradiated WT fish received 0.2 million KM blood cells from donors and were then raised under normal conditions. All four surviving recipients of Tg(hsp70:p210 BCR/ABL1 -lyz:DsRed) CMLlike donor cells developed CML-like disease within 2-3 weeks of transplantation with whole KM blood cells, indicated by infiltration of DsRed + granulocytes into the periphery ( Figure 5A) and the robust expansion of myeloid cells in both the PB and KM ( Figure 5B). In contrast, no control fish showed signs of a CML-like phenotype. We collected leukemia cells from the PB and KM and showed that these inflated cells were BCR/ABL1 + by PCR ( Figure 5C). We concluded that the myeloid cells that accumulated in Tg(hsp70:p210 BCR/ABL1 ) fish could proliferate autonomously and could cause CML-like disease in a WT host.

Tg(hsp70:p210 BCR/ABL1 ) transgenic leukemic model responded to chemotherapeutic drug treatment
Recent studies demonstrated that zebrafish shares 82% of disease-associated targets and numerous drug metabolism pathways with humans. 12 To determine if the pharmacological mechanism in Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish was also conserved compared with CML patients, we treated the WT and Tg(hsp70:p210 BCR/ABL1 ) embryos with the widely used anti-CML drugs, imatinib, dasatinib, and bosutinib, to the maximum teratogenic doses, with DMSO as a placebo (Online Supplementary   A C E D B Figure S4). After incubation with these TKI for 48 h, we calculated the numbers of lcp1 + myeloid cells in WT and Tg(hsp70:p210 BCR/ABL1 ) larvae in the posterior blood island (PBI) region at 5 dpf ( Figure 6A and B). All the TKI significantly reduced the number of lcp1 + myeloid cells in Tg(hsp70:p210 BCR/ABL1 ) larvae compared with the DMSO control group. In addition, lower concentrations (20 and 40 mmol/L) of imatinib significantly reduced the expanded lcp1 + myeloid population in Tg(hsp70:p210 BCR/ABL1 ) larvae, but the number of lcp1 + myeloid cells was also significantly reduced in WT larvae at higher concentrations (80 mmol/L) compared with DMSO (Online Supplementary Figure S5). These results suggest that high doses of imatinib may affect normal myelopoiesis, which may be associated with more adverse events or unpredictable offtarget effects. Further studies are needed to clarify these effects and to support the clinical treatment of patients with CML.

Discussion
We constructed a new germline of transgenic zebrafish expressing the hBCR/ABL1 fusion protein. Expression of hBCR/ABL1 in Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish altered hematopoiesis by up-regulating myeloid genes, as detected in larvae at 3 dpf. Adult Tg(hsp70:p210 BCR/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 line 21 established by Koschmieder et al. in 2005. However, these mice only survive for 4-17 weeks, while adult Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish could survive 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:p210 BCR/ABL1 ) transgenic model was increased by hBCR/ABL1 heat shock. This Tg(hsp70:p210 BCR/ABL1 ) transgenic model may thus provide insights into the mechanism that drives the transition 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:p210 BCR/ABL1 ) embryos, suggesting that the pharmacological pathways in this model were similar to those in human CML. The discovery of imatinib has greatly improved the longevity and quality of life of patients with CML; however, some patients develop 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 remission. 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 combating CML. Further studies are planned to generate sitedirected mutations of the ABL1 kinase domain in Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish using gene-editing technology (such as CRISPR/Cas9). The ABL1 kinase domain is frequently mutated in clinical cases, and examination 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    [27][28][29] The current results show that icaritin could reduce the expanded lcp1 + myeloid population in Tg(hsp70:p210 BCR/ABL1 ) embryos, consistent with these previous findings. Overactivation of PI3K/AKT/mTOR is known to play a pivotal role in many human cancers, thus providing strong support for the therapeutic anticancer application of PI3K/Akt/mTOR inhibitors. 30,31 Sadovnik et al. found that escape of CML LSC was disrupted by the addition of PI3K/mTOR blockers. 32 Furthermore, the PI3K/mTOR dual inhibitor BEZ235 had beneficial effects on a variety of tumors in vivo and in vitro, including lymphoid malignancies 33 and myeloid malignancies. 34, 35 Bendell also identified the active-site inhibitor CC-223, which targets both mTORC1 and mTORC2 through mTOR kinase activity to inhibit activation of AKT and 4EBP1, as a promising therapeutic agent with activity against many non-Hodgkin lymphoma and solid tumor cell lines. 36 In the current study, the Tg(hsp70:p210 BCR/ABL1 ) embryonic zebrafish model responded well to both BEZ235 and CC-223. Overall, these results suggest that targeting the PI3K/Akt/mTOR signaling pathway may be an effective strategy for overcoming CML therapy resistance. Unexpectedly, however, Tg(hsp70:p210 BCR/ABL1 ) larvae did not respond well to the sphingosine 1-phosphate antagonist, FTY720, and the number of lcp1 + myeloid cells in WT zebrafish conversely increased after treatment with FTY720. Previous studies reported that the FTY720-mediated PP2A reactivation could markedly reduce the survival and self-renewal of CML-quiescent HSC through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and βcatenin. 37 We therefore hypothesized that, although sphingosine 1-phosphate may play a role in hematopoietic regulation, further studies are needed to determine its precise mechanism. LY364947, 38,39 ciliobrevin A, 40 DB07268, 41 selonsertib, 42 NQDI-1, 43 AZD3759, 44 and icotinib 45 have recently been shown to target key factors and signaling pathways essential for the survival of CML LSC and other CSC, including transforming growth factor β, 39 Hedgehog, 46 c-Jun N-terminal kinases, 47 apoptosis signalregulating kinase 1, 48 and epidermal growth factor receptor. 49 The Tg(hsp70:p210 BCR/ABL1 ) transgenic zebrafish embryos in the current study also responded well to these compounds. Our findings, therefore, suggest that inhibition of BCR/ABL1 kinase-dependent or kinase-independent pathways ( Figure 6D) might offer potential for overcoming resistance to TKI and thus eradicate LSC, thereby paving the way for the development of novel, more effective LSC-eradicating treatment strategies for CML.

A C D B
the protein functions, pharmacological mechanisms, and toxicology of novel targeted drugs, thus improving the cure rate for patients with CML.