Distinct EphB4-mediated mechanisms of apoptotic and resistance to dasatinib in human chronic myeloid leukemia and K562 cell lines

Leukemia Research 63 (2017) 28–33

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Research paper

Distinct EphB4-mediated mechanisms of apoptotic and resistance to dasatinib in human chronic myeloid leukemia and K562 cell lines Wei-Hong Zhaoa,1, Bin-Tao Huangb,

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MARK

, Jian-Yu Zhangc,1, Qing-Chun Zengd

a

Department of Gastroenterology, The Affiliated Hospital of Inner Mongolia Medical University, 010059, Hohhot, China Department of Hematology, The Affiliated Hospital of Inner Mongolia Medical University, 1 TongDao Avenue North, 010059 Hohhot, China c College of Basic Medical Science, Inner Mongolia Medical University, Hohhot, Inner Mongolia, 010110, China d Department of Cardiology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Ave, Guangzhou, Guangdong, 510515, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Chronic myeloid leukemia EphB4 RhoA EphrinB2

Objective: To determine the role and mechanism of EphB4 in dasatinib (DAS) resistance in advanced chronic myeloid leukemia (CML), we explored the EphB4-mediated apoptotic and matrix microenvironment pathway in human CML and K562 cell lines. Method: Heparinized bone marrow samples were obtained from enrolled five patients (identified as A to E and visits identified by number) at initial diagnosis (A1–E1) and in the DAS-resistance advanced phase (A2–E2). Meanwhile, highly DAS-resistant cells, named K562-R cells, were obtained from K562-W cells with increasing concentrations of DAS. Stable under-expressing EphB4 cells (K562-R-EphB4-sh) were obtained from K562-R cells by RNA interference. K562-W, K562-R and K562-R-EphB4-sh cells (108) were respectively injected subcutaneously on the dorsal surface of BALB/C female nude mice to establish the xenografts models. Result: The mRNA/protein of EphB4 was overexpressed in the DAS-resistant A2–E2 in comparison with the A1–E1 human cell lines. Further, compared with K562-R cells, the expressions of EphB4 and p-Rac1/Cdc42 protein/mRNA were significantly downregulated in K562-R-EphB4-sh cells (P < 0.01). K562–R cells showed the highest DAS resistance (IC50 10.54 ± 0.67 μg/ml), but K562-R-EphB4-sh cells became sensitive to DAS (IC50 1.02 ± 0.1 μg/ml, P < 0.01). The expression of EphB4/p-RhoA/MCL-1 protein was gradually increased in the stimulating of EphrinB2-Fc, which partly made K562-R-EphB4-sh cells restore sensitivity to DAS (4.18 ± 0.30 μg/ml). Meanwhile, the K562-R-EphB4-sh xenografts group had relatively good efficacy compared to K562-R xenografts nude mice receiving the same dose of DAS. The analysis of xenografts tissue also suggested parallel results with the overexpression of EphB4/RhoA/ROCK1/PTEN/MCL-1 in K562-R xenografts, which decreased in the A2-R-EphB4-sh xenografts (P < 0.01). Conclusions: The present study found that a new DAS resistance pathway of EphB4 overexpression was triggered by EphrinB2-Fc, which induced the resistance to DAS by activating RhoA/ROCK1/PTEN/MCL-1 signaling.

1. Introduction Most chronic myeloid leukemia (CML) patients in a chronic phase treated with imatinib (IM) have been well controlled; however, some patients still relapse and/or progress to an accelerated phase or blast crisis [1]. Presently, the new second generation tyrosine kinase inhibitors (such as dasatinib) have been proven effective in halting the oncogenic activity of most BCR-ABL mutants. Dasatinib (DAS), as currently implemented into the first line of therapy, is 300 times more potent than IM at BCR-ABL inhibition and has few side effects [2,3]. An abnormal bone marrow (BM) microenvironment, which provides

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a protective environment for leukemia stem cells [4] make leukemia cells remit chemotherapy, become resistant to drugs and thereby relapse. This is called bone marrow matrix microenvironment-mediated drug resistance. Mutations in the kinase domain of BCR-ABL are the most prevalent mechanisms of acquired kinase inhibitor resistance in patients with CML. However, some mechanisms (such as matrix microenvironments) of drug-resistance concerning a non-kinase domain of BCR-ABL are concerning. The Rho-associated coiled-coil containing protein kinase (ROCK) serine/threonine kinase is a major downstream effector of Rho GTPases that mediates membrane blebbing, enhances actin–myosin contraction,

Corresponding author. E-mail address: [email protected] (B.-T. Huang). These authors equally contributed to this work and should be considered as co-first authors.

http://dx.doi.org/10.1016/j.leukres.2017.10.014 Received 8 August 2017; Received in revised form 26 September 2017; Accepted 26 October 2017 Available online 27 October 2017 0145-2126/ © 2017 Elsevier Ltd. All rights reserved.

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sensitivity to DAS, and resistant K562 cells were labeled as K562-R. K562-R-EphB4-sh represented the established DAS resistant cells with EphB4 knockdown by shRNA from our previous study [10].

and activates caspase signaling cascades and cellular apoptosis [5] in the matrix microenvironment. Meanwhile, as a main regulatory factor promoting the stability of the microenvironment [6], EphB4 (erythropoietin-producing hepatocyte receptor B4), a member of the largest receptor protein tyrosine kinase family, plays an important role in tumorigenesis and regulates diverse cell functions [7], which promote tumor-adhesion-mediated drug resistance via interplay with the extracellular matrix and modulation of Rho family members [8]. As a nonkinase resistance mechanism comes from the matrix microenvironment, recent findings have shown that EphB4/EphrinB2 signaling may be involved in the pathology of leukemia [9], and over expression of EphB4 is associated with IM resistance through the RhoA mechanism in CML [10,11]. DAS was proven to be effective in the treatment of IM or/and nilotinib-resistant CML patients, especially in both chronic-phase (CP) and accelerated-phase (AP) cohorts [12]. However, fewer patients with advanced CML can reach stable remission and have to receive allo-SCT. Most patients with IM- or/and nilotinib-resistant CML still gain DASresistance in the treatment of DAS. Furthermore, it was not determined whether EphB4/EphrinB2 plays an important role in the survival and resistance to DAS chemotherapy in advanced CML. To determine the role and mechanism of EphB4 in DAS resistance to advanced CML, we investigated the role of EphB4 and explored the EphB4-regulated RhoA molecule signals in human CML and K562 cell lines.

2.4. K562 xenografts model BALB/C female nude mice (four to five weeks of age; SLRC laboratory animal center, China) were used in xenografts experiments. K562-W, K562-R, and K562-R-EphB4-sh cells (108) were injected subcutaneously on the dorsal surface of female nude mice. The tumor volume was calculated as width2 × length × l/2, as measured with a digital caliper. All animal experiments were conducted according to the policy and procedures provided by the Committee for Animal Research and Ethics and were approved by the Ethics Committee of the Inner Mongolia Medical University. 2.5. Oral DAS therapy regimen of nude mice with xenografts According with the standards of xenografts models, BALB/C nude mice began oral DAS by perfusion. In the accelerated and blastic phases, as well as in leukemia, 70 mg twice daily is recommended [13]. Based on the dose of human CML (body weight 60 kg), the oral dose of mice was calculated as 70/60 × 9.1 = 10.6 ug/g body weight, twice, for 30 days. The volume of the xenografts and the weights of the mice were recorded during the treatment of 30 days.

2. Materials and methods 2.6. Semi-quantitative PCR (SQ-PCR) analysis 2.1. Patients and cell culture All cell lines from patients or K562 used in experiments were harvested in the exponential growth phase. Total RNA was isolated with TRIzol reagent (Invitrogen, USA). SQ-PCR was performed on ABI 9700 system. Primers for gene amplification were designed with Primer 5.0 and sequences are as follows: EphB4-upstream primer: 5′-ACTCCTTCCTGCGGCTAA-3′; EphB4-downstream primer: 5′-AGACGAGGTTGCTGTTGACT-3.

Five enrolled (Bcr/abl, P210) CML patients (age range from 30 to 65 years old) initially received IM and achieved continued major molecular response, but refractory/accelerated 2–5 years later. Those harboring highly-resistant mutations (T315I/A, F317L/V/C and V299L) had not been detected. Then, the patients continued to receive DAS treatment for one year, but they still did not enter hematological remission. Heparinized BM samples were obtained from enrolled five patients with informed consent in compliance with the Declaration of Helsinki at initial diagnosis and in the DAS-resistance advanced phase. The cells were cultured in a RPMI-1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C and a humidified atmosphere of 5% CO2. Continuously growing cells were obtained from BM samples at first diagnosis and in the advanced phase of DAS-resistance and were named A1–E1 and A2–E2, respectively.

2.7. Western blot analysis

K562 wild type cells (K562-W, American Type Culture Collection, Manassas, VA, USA) were incubated with DAS (initial at 0.05 mg/ml) to establish DAS resistance using the step-wise method described previously [10]. We cultured K562-W cells with increasing concentrations of DAS to generate highly resistant cell. The gradient concentrations of DAS (0.05 ∼ 10.0 μg/ml) were added. DAS-resistant cell line from K562-W, named K562 resistance type cell (K562-R), were obtained after 10 months and were maintained in the presence of 5 μg/ml DAS.

Cells were washed in PBS at 4 °C and total proteins were isolated by RIPA lysis buffer. After removing the nude mice subcutaneous mass, xenografts tissue proteins were also isolated by RIPA lysis buffer (50 mg: 1 ml). Proteins (80 μg) were separated by SDS-PAGE and transferred onto PVDF membranes with the Bio-Rad transblot system (Bio-Rad, Hercules, CA, USA). After blocking in 5% bovine serum albumin (BSA) for two hours, membranes were incubated overnight in a primary antibody diluted in 5% BSA at 4 °C. The following primary antibodies were used: antiEphB4 (R & D, USA), anti-Phospho-Rac1/cdc42, anti-ROCK1, antiPhospho-PTEN, anti-Mcl-1 (Cell Signaling Technology, USA), and antiβ-actin (Santa Cruz Biotechnology, USA). The membranes were incubated for one hour with HRP-conjugated cow anti–goat (or rabbit) immunoglobulin (1/5000) (from Santa Cruz Biotechnology, USA), diluted in 5% BSA. After washing, the enhanced chemiluminescence kit (Santa Cruz Biotechnology, USA) was applied and positive bands were detected on X-ray films. All of the experiments were repeated three times and showed similar results.

2.3. Knockdown of EphB4 by RNA interference (RNAi) and CCK8 assay

2.8. Statistical analysis

According the previous study, oligonucleotides for shRNA were designed using ABI online software. The stable underexpressing EphB4 cells (K562-R-EphB4-sh) represented the established DAS-resistant K562-R cells with EphB4 knockdown by shRNA from our previous study procedure [10]. GFP-positive cells (68%) were sorted after infection by flow cytometry. Then, stable underexpressing EphB4 of cell lines from K562-R were obtained (K562-R-EphB4-sh). Cells were re-examined for

Statistical analysis was carried out using SPSS 13.0 statistical software. Values represented the mean of three independent experiments and were expressed as mean ± SD. The expressions of protein/mRNA were evaluated by ANOVA or T-test analysis. A paired t-test was used in the comparison of xenografts volumes by oral DAS intervention. OS of K562 xenografts nude mice were estimated by the Kaplan-Meier method. P value < 0.05 was considered statistically significant.

2.2. Induction of K562-R cells with high DAS-resistance

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Fig. 3. Expressions of EphB4 and Rac1/cdc42 mRNA in K562 cell lines. Expressions of EphB4 and Rac1/cdc42 mRNA transcripts were compared by reverse transcription-PCR among K562-W, K562-R, and K562-R-EphB4-sh cell lines. Band intensity on gel electrophoresis was quantified using computer software. The experiments were repeated three times and showed similar results.

regarded K562 (including K562-W, K562-R and K562-R-EphB4-sh) cells as main subjects in the DAS-resistant experiment. The EphB4 mRNA and protein were overexpressed in the K562-R cells in comparison with the K562-W cells. But the expressions of EphB4 mRNA and protein significantly decreased in knockdown K562-R-EphB4-sh cells compared with K562-R and K562-W (Figs. 2 and 3, P < 0.01) cells. Then the K562-R-EphB4-sh cell lines were well established. We also examined the mRNA and phosphorylation protein of RhoA molecules (Rac1/ Cdc42) in K562 cells. The mRNA/phospho-protein of Rac1/cdc42 was significantly increased in K562-R cells but significantly decreased in K562-W and K562-R-EphB4-sh cells (Figs. 2–3, P < 0.01). At the same time, the K562-R-EphB4-sh cell was co-incubated with EphrinB2-Fc (4 μg/ml), and IgG-Fc (0.4 μg/ml) as the control for 12 and 24 h. The expression of EphB4/p-Rac1/Cdc42/MCL-1 was gradually increased in the EphrinB2-Fc group but continuously downregulated in the IgG-Fc control group from 12 to 24 h (P < 0.01, Fig. 4). In the control group of IgG-Fc, there were no differences in the expression of EphB4/p-Rac1/Cdc42 and MCL-1 for 0, 12 and 24 h (P > 0.05, Fig. 4). This demonstrated that the EphB4/EphrinB2 signal protein regulated the p-RhoA/MCL-1 activations in the K562 cells. Meanwhile, the resistance testing also showed there were significant differences in IC50 for K562 cells (P < 0.01). K562–R cells showed the highest DAS resistance (IC50 10.54 ± 0.67 μg/ml), and K562-REphB4-sh cell became partly sensitive to DAS (IC50 1.02 ± 0.1 μg/ml) compared to K562-R cells (P < 0.01, Table 1). Further, K562-REphB4-sh cells were in co-incubation with EphrinB2-Fc (4 μg/ml) for 24 h. CCK-8 results showed an IC50 of 4.18 ± 0.30 μg/ml for the EphrinB2-Fc group, which were significantly lower than in the K562-R cells group (P < 0.01, Table 1). The result showed that the expression of EphB4 significantly increased in K562-R cells with high DAS resistance, but decreased in K562-R-EphB4-sh cells with relatively low DAS resistance. EphrinB2-Fc partly made K562-R-EphB4-sh cells restore sensitivity to DAS. It indicated that the EphB4/EphrinB2 contributed to DAS resistance at the level of cell level study.

Fig. 1. Expressions of EphB4 protein/mRNA in A1–E1 and A2–E2 human cell lines. Expression of EphB4 proteins were analyzed by Western Blotting among A1–E1, and A2–E2. Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes with the Bio-Rad Transblot system, and detected with the indicated antibodies. Expressions of EphB4 mRNA transcripts were compared by reverse transcriptionPCR among A1–E1, and A2–E2. Band intensity on gel electrophoresis was quantified using computer software. The experiments were repeated three times and showed similar results.

3. Results 3.1. The expressions of EphB4 mRNA/protein increased in DAS-resistant human CML cell lines The previous study showed that the EphB4 mRNA and protein were overexpressed in the IM-resistant human CML cell lines compared with the IM-sensitive cell lines [10]. In the experiment, PCR and Western blot were still used to detect the expression of the EphB4 mRNA and protein in five patients with CML at the first diagnosis (A1–E1) and in the DAS-resistant advanced phase (A2–E2). The mRNA and protein of EphB4 were overexpressed in the DAS-resistant A2–E2 human CML cell lines in comparison with the A1–E1 human CML cell lines (P < 0.001, Fig. 1). It meant that EphB4 also played an important DAS resistance role in advanced CML patients. 3.2. EphB4/RhoA regulated DAS resistance in K562 cell lines Previous data indicated that EphB4/RhoA overexpression was associated with DAS resistance in human CML cell lines [10]. Further, we

3.3. Establishing the K562 xenografts models Twenty-two days after implantation, the tumors reached an average size of over 400 mm3. There were no differences (P > 0.05, Table 2) in average volume among K562-W (448.60 ± 31.94 mm3), K562-R (459.79 ± 31.62 mm3), and K562-R-EphB4-sh (446.13 ± 22.40 mm3) xenografts. Furthermore, the xenografts models were determined by histopathology. It clearly showed dense cells, blue hyperchromatic nuclei, and tissue stroma in the xenografts histopathology section by HE dying (Fig. 5). There were obvious green fluorescent reactions in the cell nuclei of the K562-R-EphB4-sh xenografts but none in the K562-W and K562-R xenografts under the fluorescence microscope. This showed that the K562-

Fig. 2. Expressions of EphB4 and Rac1/cdc42 protein in K562 cell lines. Expression of EphB4 and Rac1/cdc42 proteins were analyzed by Western Blotting among K562-W, K562-R, and K562-R-EphB4-sh cell lines. Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes with the Bio-Rad Transblot system, and detected with the indicated antibodies. The experiments were repeated three times and showed similar results.

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Fig. 4. Expressions of EphB4/RhoA/MCL-1 in K562-R-EphB4-sh/ EphrinB2-Fc or IgG-Fc cell groups. K562-R-EphB4-sh cells were in co-incubation with EphrinB2-Fc (4 μg/ml) for 24 h, and IgG-Fc (0.4 μg/ml) was the control. Expressions of EphB4/ RhoA/MCL-1 were analyzed by Western Blotting proteins in K562-REphB4-sh and EphrinB2-Fc cell groups, respectively. Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes with the Bio-Rad Transblot system and detected the indicated antibodies. The experiments were repeated three times and showed similar results.

R-EphB4-sh xenografts were well established (Fig. 5).

Table 2 Volumes of K562 xenografts after injecting cell from subcutaneous.

3.4. K562-R-EphB4-sh xenografts tumor was sensitivity to DAS Three groups (N = 6/group) of nude mice received an oral dose of DAS (10.6 ug/g of body weight twice for 30 days) treatment after the xenografts were established. K562-R xenografts volumes continued to increase after receiving DAS treatment (459.79 ± 31.62 mm3 versus 610.43 ± 44.43 mm3, P < 0.01); however, no significant differences in K562-W (448.60 ± 31.94 mm3 versus 434.99 ± 68.30 mm3, P > 0.05) and K562-R-EphB4-sh (446.13 ± 22.40 mm3 versus 449.49 ± 67.35 mm3, P > 0.05) xenografts volumes with DAS treatment were found (Table 3). Meanwhile, all of the nude mice with a K562-R xenografts tumor died. Three and four nude mice died separately in the K562-W and K562-R-EphB4-sh xenografts groups during the period of treatment (30 days). As a result, the K562-R-EphB4-sh and K562-W xenografts groups had better efficacy receiving oral DAS than K562-R xenografts nude mice receiving the same dose of DAS. Obviously, K562-W and K562-R-EphB4-sh xenografts nude groups had more OS advantages than the K562-R xenografts nude group (median: 26 days, 15 days versus 9 days, Fig. 6, P < 0.01).

N = 6/group

Volume of xenografts(Mean ± SE, mm3)

Days after injection

7 days

14 days

22 days

K562-W K562-R K562-R-EphB4-sh P value

153.46 ± 22.77 163.29 ± 19.42 138.71 ± 17.24 > 0.05

210.66 ± 20.10 215.70 ± 18.44 184.30 ± 12.46 > 0.05

448.60 ± 31.94 459.79 ± 31.62 446.13 ± 22.40 > 0.05

resistant or intolerant CML-CP, and inhibit all tested IM-resistant mutations, except T315I [14]. Compared with nilotinib, DAS not only inhibits BCR-ABL and SRC-family kinases, but also c-KIT, PDGFR-alpha and beta, and Ephrin receptor kinases [15]. It has been suggested [10] that the overexpression of EphB4 is a new marker of IM resistance. In the findings, the determined five subjects (A–E) with resistance to DAS were not detected as having these harboring highly-resistant mutations (T315I/A, F317L/V/C and V299L). The expression of EphB4 significantly increased in A2–E2 human cells with high DAS resistance but decreased in A1–E1 human cells with sensitivity to DAS. Further, the expression of EphB4 was also significantly increased in K562-R cells and xenografts but decreased in K562-R-EphB4-sh cells and xenografts with underexpressing EphB4. Meanwhile, K562-REphB4-sh cells became recovery sensitive to DAS, and K562-R cells showed high DAS resistance. The K562-R-EphB4-sh and K562-W xenografts group had relatively good efficacy receiving oral DAS compared with K562-R xenografts nude mice receiving the same dose of DAS. As a result, EphB4 has a definite resistance to DAS on the level of the K562 cells and xenografts study. The introduction of tyrosine kinase inhibitors (TKIs)-IM has fundamentally changed the management of CML. However, once the disease has progressed to an accelerated or blast phase, there is no consensus regarding optimal therapy [16]. Presently, DAS is approved for the treatment of patients with BCR-ABL-positive CML who are resistant or intolerant to IM in the chronic, accelerated, and blast phases. The previous report showed [10] that the overexpression of EphB4 is associated with IM resistance through the RhoA mechanism in CML. It is an important pathway inducing apoptosis and mitochondrial injury for activating RhoA/ROCK1/PTEN signaling in human leukemia cells [17]. Our study found that the expressions of p-Rac1/Cdc42, ROCK1,

3.5. The molecule mechanism of DAS resistance in K562 xenografts Meanwhile, an analysis of the xenografts tissue suggested parallel results with the high EphB4 expression in K562-R xenografts and the low expression in K562-R-EphB4-sh and K562-W xenografts (P < 0.01, Fig. 7). Accordingly, the expressions of phosphorylation Rac1/Cdc42 and MCL-1 were increased in K562-R xenografts but decreased in K562R-EphB4-sh and K562-W xenografts (P < 0.01, Fig. 7). In order to determine the roles of ROCK1/PTEN, the findings showed that phospho-PTEN and ROCK1 were significantly increased in the high EphB4-expression K562-R xenografts but decreased in K562-R-EphB4sh xenografts (P < 0.01, Fig. 7). 4. Discussion DAS and nilotinib, as the second potent, oral generation TKIs, bind to both the active and inactive conformation of the ABL1 kinase domain, have been found to be effective in treating patients with IM Table 1 DAS resistance to change for K562 cells (Mean ± SE). N = 3/group

K562-W

K562-R

K562-R-EphB4-sh

K562-R-EphB4-sh/EphrinB2-Fc

P

IC50(μg/ml)

0.11 ± 0.00

10.54 ± 0.67

1.02 ± 0.10

4.18 ± 0.30

< 0.01

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Fig. 5. Tissue morphology from xenografts modes. Light microscope observation of K562-W, K562-R, and K562-REphB4-sh xenografts tissues were completed by HE dying (100×). Fluorescence microscope observation of K562-W, K562-R, and K562R-EphB4-sh xenograft tissues by GFP-positive label (100×). There was obvious green fluorescent reaction in the cell nuclei of K562-REphB4-sh xenograft, but none in K562-W and K562-R xenografts revealed by fluorescence microscope.

EphrinB2-Fc and induced resistance to DAS by activating RhoA/ ROCK1/PTEN/MCL-1 signaling.

Table 3 Volume change (Mean ± SE, mm3)of K562 xenografts by DAS therapy. N = 6/group

Before treatment

After tratment

P value

K562-W K562-R K562-R-EphB4-sh P value

448.60 ± 31.94 459.79 ± 31.62 446.13 ± 22.40 > 0.05

434.99 ± 68.30 610.43 ± 44.43 449.49 ± 67.35 < 0.01

> 0.05 < 0.01 > 0.05

Conflict of interest All authors declare that they have no conflict of interest. I would like to declare on behalf of my co-authors that this work is original research that has not been published previously and is not under consideration for publication elsewhere, either in part of in whole. All the authors listed have approved the manuscript that is enclosed.

phospho-PTEN, and MCL-1 were increased in K562-R cells and xenografts but decreased in K562-R-EphB4-sh cells and xenografts. The EphB4/RhoA overexpression induced resistance to DAS by activating the ROCK1/PTEN/MCL-1 mechanism. EphB4/Ephrins play important roles in oncogenesis and in tumor growth progression [18,19]. Previous research demonstrated that EphB4/Ephrins promoted cell adhesion and mediated drug resistance through contact with the extracellular matrix and regulating Rho family members in the microenvironment [20]. In the study, the EphrinB2-Fc made K562-R-EphB4-sh cells restore sensitivity to DAS by stimulating the expression of EphB4. Accordingly, in the stimulation of EphrinB2Fc, the expressions of EphB4/RhoA/MCL-1 were significantly increased in K562-R-EphB4-sh cells. In summary, the present study found that a new DAS resistance pathway of EphB4 overexpression was triggered by

Funding This study was funded by the National Natural Science Foundation of China (grant number 81460027). Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/ or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The animal experiments were conducted according to the policy and procedures provided by the Committee for Animal Research and Ethics and were Fig. 6. OS of K562 xenografts nude mice. OS of K562-related (including K562-W, K562-R, and K562-R-EphB4-sh) xenografts nude mice were estimated by the Kaplan-Meier method after receiving the same dose of DAS (N = 6/group).

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[2] [3]

[4] [5] [6] [7]

[8]

[9] [10]

[11] Fig. 7. Expressions of EphB4/RhoA/ROCK1/PTEN/MCL-1 signal protein in K562 xenografts tissues. The expressions of signal protein were analyzed by Western Blotting proteins in xenografts tissues. Phosphorylations of Rac1/cdc42 and PTEN were detected by phosphospecific antibodies. ROCK1 and MCL-1 were detected by anti-ROCK1 and anti-Mcl-1 antibodies. The experiments were repeated three times and showed similar results.

[12]

[13] [14]

approved by the Ethics Committee of the Inner Mongolia Medical University.

[15] [16]

Informed consent [17]

Informed consent was obtained from the individual participant included in the study.

[18]

Acknowledgments

[19]

We would like to thank the patient and the clinicians from the participating sites.

[20]

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