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MXD1 regulates the imatinib resistance of chronic myeloid leukemia cells by repressing BCR-ABL1 expression
Accepted Manuscript Title: MXD1 regulates the imatinib resistance of chronic myeloid leukemia cells by repressing BCR-ABL1 expression Authors: Chen Huan, Lou Jin, Wang Heng, An Na, Pan Yuming, Du Xin, Zhang Qiaoxia PII: DOI: Reference:
S0145-2126(18)30446-6 https://doi.org/10.1016/j.leukres.2018.10.012 LR 6062
To appear in:
Leukemia Research
Received date: Revised date: Accepted date:
27-6-2018 8-10-2018 24-10-2018
Please cite this article as: Huan C, Jin L, Heng W, Na A, Yuming P, Xin D, Qiaoxia Z, MXD1 regulates the imatinib resistance of chronic myeloid leukemia cells by repressing BCR-ABL1 expression, Leukemia Research (2018), https://doi.org/10.1016/j.leukres.2018.10.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page Title: MXD1 regulates the imatinib resistance of chronic myeloid leukemia cells by repressing BCR-ABL1 expression Author names: Chen Huan, Lou Jin, Wang Heng, An Na, Pan Yuming, Du Xin *, and Zhang Qiaoxia * Author affiliations: Shenzhen Bone Marrow Transplantation Public Service Platform,
Affiliated Hospital of Shenzhen University, Shenzhen, China
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Shenzhen Institute of Hematology, Shenzhen Second People's Hospital, The First
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Corresponding author: Zhang Qiaoxia, [email protected], 13925263107 Present address: NO. 3002 Sungang West Road, Futian District, Shenzhen City, Guangdong Province, China
Transcriptional repressor MXD1 shows low expression in CML patients and imatinib-resistant K562/G01 cell line
Overexpression of MXD1 inhibits the proliferation of K562 cells and sensitize
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K562/G01 cell line to imatinib
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Highlights
MXD1 downregulates BCR-ABL1 expression by inhibiting the transcriptional
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Abstract
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activity of the BCR-ABL1 gene promoter.
Tyrosine kinase inhibitors have achieved unprecedented efficacy in the treatment
of chronic myeloid leukemia (CML); however, imatinib resistance has emerged as a
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major problem in the clinic. Because the overexpression of BCR-ABL1 critically contributes to CML pathogenesis and drug resistance, targeting the regulation of BCR-ABL1 gene expression may be an alternative therapeutic strategy. In this study, we found that the transcriptional repressor MXD1 showed low expression in CML patients and was negatively correlated with BCR-ABL1. Overexpression of MXD1 markedly inhibited the proliferation of K562 cells and sensitized the imatinib-resistant
K562/G01 cell line to imatinib, with decreased BCR-ABL1 mRNA and protein expression. Further investigation using reporter gene analysis showed that MXD1 significantly inhibited the transcriptional activity of the BCR-ABL1 gene promoter. Taken together, these data show that MXD1 functions as a negative regulator of BCR-ABL1 expression and subsequently inhibits proliferation and sensitizes CML
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cells to imatinib treatment.
Keywords: Chronic myeloid leukemia, MXD1, BCR-ABL1, Tyrosine kinase inhibitors,
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Drug resistance
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1. Introduction
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Chronic myeloid leukemia (CML) is a common myeloproliferative neoplasm
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characterized by the chromosomal translocation t(9;22) (q34;q11) [1]. This translocation causes the generation of a fusion oncogene, namely, the BCR-ABL1
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encoding fusion protein with constitutive tyrosine kinase activity, which gives rise to
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the uncontrolled growth of myeloid cells in the bone marrow through a series of downstream pathways [2,3]. One of the first-line treatments for CML is imatinib, a tyrosine kinase inhibitor (TKI) that competitively binds to the ATP-binding site of
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Bcr-Abl and blocks the downstream signal pathway [4,5]. TKIs have dramatically improved the overall survival rate of CML patients [6,7]. However, TKI resistance has
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become a major clinical problem for CML, mainly as a consequence of BCR-ABL1 mutations, BCR-ABL1 overexpression and other BCR-ABL1 independent pathways [8,9]. Bcr-Abl protein abundance heavily contributes to CML pathogenesis and drug
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resistance. Additionally, the expression level of BCR-ABL1 significantly influences the generation of BCR-ABL1 mutations [10,11]. One suggestion is that targeting Bcr-Abl fusion protein expression might override aberrant Abl kinase activity. However, the molecular mechanism of BCR-ABL1 gene regulation needs to be further characterized. MAX dimerization protein 1 (MXD1) is a transcription factor that belongs to the
MYC/MXD/MAX
family,
whose
members
typically
contain
a
basic
helix-loop-helix-leucine-zipper (bHLHZip) domain [12]. With this domain, MXD1 binds to the E-box DNA element in the promoters of target genes to regulate their transcription. As a transcriptional repressor, MXD1 mainly influences cell transformation, proliferation, apoptosis, and differentiation [13,14,15]. MXD1 has been reported to be an antagonist of MYC that inhibits cell proliferation and
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antagonizes the transforming activity of MYC [16]. MXD1 is also involved in the survival, migration and invasion of cancer cells, such as pancreatic cancer cells,
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gastric cancer cells and lung cancer cells [17,18,19]. To date, the role of MXD1 in CML has not been clarified. Since MXD1 E-box consensus binding sites are present in
the promoter of BCR-ABL1 [20], we aimed to determine whether MXD1 regulates the
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expression of BCR-ABL1.
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In this study, we investigated the expression and biological function of MXD1 in
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CML patients, K562 cells and imatinib-resistant K562/G01 cells. The results showed that the level of MXD1 was significantly lower in CML patients and K562/G01 cells
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than in controls. In addition, MXD1 inhibited the proliferation and enhanced the
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imatinib sensitivity of K562 cells and K562/G01 cells by reducing BCR-ABL1 gene expression.
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2. Materials and Methods
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2.1. Cell lines and human samples Human CML cell lines K562 and K562/G01 (imatinib induced resistance) were
purchased from the Shanghai Institute of Life Sciences (Shanghai, China) and State
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Key Laboratory of Experimental Hematology (Tianjing, China), respectively. Cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37˚C in 5% CO2. To maintain drug resistance, imatinib (1 µg/ml) was applied to K562/G01 cells at least 2 weeks before experiments. The peripheral blood cells of CML patients and healthy donors were collected at Shenzhen Second People’s Hospital. In total, 91 cases of CML patients and 55 cases
of normal control were enrolled. All CML patients had clinical data on their BCR-ABL1 (p210) levels, shown as the percentage of p210 to ABL as measured by qRT-PCR. All participants signed informed consent forms, and all studies involving the use of human blood were performed in accordance with the Declaration of Helsinki. This study was approved by the Ethics Committee of Shenzhen Second
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People’s Hospital.
2.2. RNA isolation and quantitative real-time PCR analysis
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Total RNA was extracted from cultured cells with RNAiso Plus (Takara, JAP). Reverse transcription into cDNA was carried out with the PrimeScriptTM RT reagent kit (Takara, JAP) according to the manufacturer’s protocol. Real-time PCR was
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performed with SYBR® Premix Ex Taq™ II (Takara, JAP). The relative expression
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level was determined with the 2-∆∆Ct method, and the human GAPDH gene was
ATTCGGGTCCAAGTG-3’
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5’-TGAACATGGTTATGCCTCCA-3’
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applied as the internal control. The primer sequences were as follows: MXD1: (forward),
5’-ACTTG
(reverse);
BCR-ABL1:
5’-TCCACTGGCCACAAAATCATACAG-3’
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5’-TCAGAAGCTTCTCCCTGACATCCG-3’ GTCAAGGCTGAGAACGGGAA-3’
(forward), (reverse);
and
(forward),
GAPDH:
5’5’-
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TCGCCCCACTTGATTTTGGA-3’ (reverse). Thermal cycling reactions were performed as follows: 95˚C for 5 min, followed by 40 cycles of two-step PCR at 95˚C
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for 5 sec and 60˚C for 34 sec.
2.3 Establishment of stably transfected cell lines
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MXD1-overexpressing lentivirus vector (pHBLV-MXD1-ZsGreen-Puro, flag) and control lentivirus vector (pHBLV-ZsGreen-Puro) were constructed and then enveloped to produce lentiviruses. After target cell infections, puromycin selection was applied to establish the stably transfected K562 cell lines. To produce lentiviruses, 293T packaging cells were cotransfected with lentivirus and packaging plasmids, namely, pSPAX2 and pMD2G, respectively. Before transfection, 293T cells were
plated in a 10-cm dish and cultured in DMEM (Gibco, USA) with 10% FBS (Gibco, USA). When the cell density reached 60~80%, 293T cells were transfected with the plasmids mentioned above. 12~16 h later, the culture medium was replaced with 5~6 ml of fresh DMEM with 10% FBS. Then, the supernatants, which contained virus, were harvested at 24 h and 48 h and concentrated by ultracentrifugation for 90 min at 50,000 g and 4°C. knock
down
MXD1,
shRNA
oligonucleotides
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(GGACCCGAATCAAGTCGACACACTA) were designed using ABI online software, the
corresponding
sequence
TTCTCCGAACGTGTCACGTAA.
of
scrambled
shRNA
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and
RNA
interference
was
vector
(pHBLV-U6-MXD1-Puro) and control lentivirus were constructed. After target cell
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infections, puromycin was added to screen positive cells with stably reduced
2.4 Cell proliferation and viability assays
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expression of MXD1.
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To test cell proliferation, a Cell Counting Kit (CCK, TransGen Biotech, China)
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was used. K562 cells were seeded into 96-well plates. Every 24 h, CCK reagents were added to each well in a 10 µl volume, the plates were incubated at 37˚C for 2 h, and the absorbance was measured at 450 nm.
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The CCK kit was also used for a cell viability assay. K562 or K562/G01 cells were seeded into 96-well plates and treated with imatinib to determine IC50 values.
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K562 cells were treated with a series of imatinib concentrations (0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 and 5.12 µM), and K562/G01 cells were treated with a series of imatinib concentrations (0, 0.5, 1, 2, 4, 8, 16, 32 and 64 µM). After 48 h, CCK
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reagents were added to each well in a volume of 10 µl, the plates were incubated at 37˚C for 2 h, and the absorbance was measured at 450 nm.
2.5 Western blot Total protein was extracted using RIPA lysis buffer (Thermos Fisher, USA) supplemented with a protease inhibitor cocktail tablet (Roche Applied Science). The
protein content was measured with a BCA protein assay kit (Thermo Fisher, USA). Protein samples (20 mg/lane) were resolved on a 10% bis-tris gel (SDS-PAGE) and transferred onto 0.22-µm PVDF membranes (Millipore, USA). After blotting, the membranes were blocked with 3% BSA in TBS-Tween for 2 h and incubated with relevant primary antibodies overnight at 4˚C. These antibodies included anti-Mxd1 (Proteintech, USA, 17888-1-AP), anti-c Abl (Cell Signaling Technology, USA,
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#2862s) and anti-Actin (Abcam, UK, ab3280). The membranes were washed three times with 1x TBS supplemented with 0.1% Tween 20, hybridized with HRP-labeled
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secondary antibodies—anti-rabbit IgG (Cell Signaling Technology, USA, #7074) and
anti-mouse IgG (Cell Signaling Technology, USA, #7076) and then visualized with a
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chemiluminescence detection reagent (Thermos Fisher, USA).
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2.6 Dual luciferase reporter assay
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pGL3-BCR promoter constructs are referred to in a previous report by Marega et al [21]. A sequence 1455 bp upstream from the ATG site of the BCR gene and several
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truncated sequences were synthesized by BGI (China). After digestion with Kpn1 and
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Xhol (Roche, USA), these sequences were inserted into the pGL3 vectors separately. 293T cells were cotransfected with pCMV3-MXD1 (Sinobiological, China) expression vectors and pGL3-BCR luciferase reporter vectors, along with pRL-TK
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(Promega, USA) vectors, by using Lipofectamine 3000 (Invitrogen, USA). Forty-eight hours after transfection, cells were harvested, and a Dual Luciferase
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Reporter Assay System (Promega, USA) was used to determine the luciferase activities according to the manufacturer’s instructions. Relative luciferase activity was
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normalized to the Renilla luciferase signal.
2.7 Statistical analysis All experiments were repeated at least three times, and data are presented as the mean ± SD. The differences were calculated using the unpaired t-test, and Spearman’s correlation was performed with GraphPad Prism 5 software. P < 0.05 was deemed statistically significant.
Results
3.1 MXD1 is downregulated in CML patients and K562/G01 cells We compared the relative levels of MXD1 mRNA in the peripheral blood cells of CML patients and healthy donors by qRT-PCR. The results showed that the MXD1
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mRNA level in CML patients was significantly lower than that in the healthy donors (0.62 ± 0.05 vs 1.25± 0.11, respectively; P < 0.001) (Fig. 1A). A further correlation
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analysis indicated that the mRNA levels of BCR-ABL1 and MXD1 were negatively correlated in CML patients (Fig. 1B). We also detected the expression of MXD1 in wild type CML cell line K562 and imatinib-resistant K562/G01 cells. The results
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showed that K562/G01 cells expressed less MXD1 mRNA and protein than K562 cells
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(Fig. 1C and D).
3.2 Overexpression of MXD1 reduces the proliferation and imatinib resistance of
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CML cells
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As the expression of MXD1 was downregulated in the CML cells, to further examine the roles of MXD1 in CML, we established a stably overexpressing MXD1 K562 cell line using recombinant lentivirus. As expected, the expression of MXD1
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was much higher in the MXD1-overexpression K562 cells (K562-MXD1) than in the control cells (K562-Con, K562 cells stably transfected with control lentivirus), as
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shown by qPCR and Western blot analyses (Fig. 2A and B). The results of the CCK assays showed that overexpression of MXD1 inhibits K562 cell proliferation (Fig. 2D). To determine the effect of MXD1 on the imatinib sensitivity of K562 cells, stably
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transfected K562 cells were treated with a series of concentrations of imatinib (0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 and 5.12 µM) for 48 h, and then, the IC50 value was calculated. The result showed that MXD1 significantly decreased the IC50 value from (0.44 ± 0.01 µM) to (0.21 ± 0.01 µM) (P < 0.001), suggesting that overexpression of MXD1 enhanced the sensitivity of K562 cells to imatinib treatment (Fig. 2E and F). To further verify the effect of MXD1 on the TKI sensitivity of CML cells, an
imatinib-resistant cell line, namely, K562/G01, was used as a study model. We added lentivirus, including MXD1 cDNA or control lentivirus, to the K562/G01 cells and used puromycin to select stably transfected cells, that is, K56/G01-MXD1 and K562/G01-Con cells. Then, the K562/G01-MXD1 cells were treated with a series of concentrations of imatinib (0, 0.5, 1, 2, 4, 8, 16, 32 and 64 µM). Overexpression of
relative to K562/G01-Con cells (4.09 ± 0.18 µM), P < 0.01 (Fig. 2G-J).
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MXD1 drastically reduced the IC50 value of K562/G01-MXD1 cells (8.20 ± 0.51 µM)
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3.3 Knockdown of MXD1 increases the proliferation and drug resistance of K562 cells
K562 cells were stably transfected with lentivirus encoding shMXD1 and
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scrambled shRNA (negative control: shNC). The shMXD1 silencing efficiency was
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determined by qRT-PCR and Western blot; MXD1 mRNA and protein levels were
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obviously decreased in K562-shMXD1 cells (79.98% and 35.58%, respectively) relative to K562-shNC cells (Fig. 2A and C). CCK assays showed that knockdown of
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MXD1 promoted the proliferation of K562 cells (Fig. 3D). However, after treatment
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with a series of concentrations of imatinib (0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 and 5.12 µM) for 48 h, the IC50 value of K562-shMXD1 cells (0.78 ± 0.05 µM) was increased significantly relative to that of K562-shNC cells (0.33 ± 0.02 µM), which
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indicated that downregulation of MXD1 increased the imatinib resistance of K562
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cells (Fig. 3E and F).
3.4 MXD1 downregulates BCR-ABL1 expression by transcriptional inhibition We have shown that BCR-ABL1 and MXD1 are negatively correlated in CML
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patients (Fig. 1B). Similarly, BCR-ABL1 mRNA and protein levels are downregulated by overexpressed MXD1 in K562 cells and in K562/G01 cells relative to negative controls (Fig. 4A-C). These results suggest that MXD1 is involved in BCR-ABL1 expression. To determine the mechanism by which MXD1 regulates BCR-ABL1, we cloned
BCR promoter constructs, with a sequence starting 1455 bp upstream of the transcriptional start site of the BCR gene, into a luciferase reporter vector and named the vector pGL3-BCR1, and 293T cells were used to conduct luciferase reporter assays. The results indicated that overexpression of MXD1 dramatically decreased the luciferase activity of pGL3-BCR1 but not pGL3-Empty (negative control), suggesting that MXD1 significantly repressed the promoter activity of BCR (Fig. 5A).
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According to Marega et al [21] and previous studies [22,23], there are two
potential regions for the transcriptional regulation of MXD1 in the BCR gene, namely,
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region1, from -1425 bp to -1196 bp, and region2, from -883 bp to -690 bp. Thus, we constructed another three vectors, as follows: pGL3-BCR2: 1455 bp upstream of the BCR coding starting site with deletion of region1; pGL3-BCR3: 1455 bp upstream of
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the BCR coding starting site with deletion of region2; pGL3-BCR4: 1455 bp upstream
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of the BCR coding starting site with deletion of both region1 and region2. The results
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showed that in the presence of MXD1, both pGL3-BCR2 and pGL3-BCR3 had reduced luciferase activity (P < 0.001). However, deletions of both region1 and
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region2 almost completely abrogated the regulation of BCR promoter activity by
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MXD1, suggesting the important roles of these two regions in the transcription of BCR
Discussion
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(Fig. 5B).
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MXD1 is an important transcriptional repressor, and the bHLHZip domain
enables MXD1 binding to a specific DNA site, namely, the CACGTG E-box element [24]. Another key structural element of MXD1 is the mSIN3 interaction domain (SID),
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which is able to recruit a mSIN3-HDAC repressor complex and then acetylates core histones [25]. Lee et al [14] demonstrated that MXD1 is a transcriptional repressor of Bcl-6, which is a major regulator of B cell neoplastic transformation, and MXD1 directly regulates Bcl-6 transcription via the E-box-like DNA sequences in the Bcl-6 upstream promoter region. Bouchard et al [26] found that in differentiating HL60 cells, MXD1/MAX complexes bind to the cyclin D2 promoter in vivo and increase
histone deacetylation, thereby regulating cyclin D2 gene expression. In summary, MXD1 can regulate the compaction and accessibility of chromatin and control the expression of different genes. Our results showed that MXD1 downregulated BCR-ABL1 expression at the mRNA and protein levels and inhibited the proliferation and increased the TKI sensitivity of CML cells.
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Although TKI imatinib has shown remarkable effects on CML therapy and improved the overall survival of patients, drug resistance and relapse tend to develop
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after long-term application [27,28]. As BCR-ABL1 levels play a critical role in its tyrosine kinase activity, drug resistance, the formation of point mutations and disease progression [8,10,29], targeting the expression of BCR-ABL1 might be an alternative
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strategy for CML treatment. Recently, several studies have shown that decreasing
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BCR-ABL1 expression represents a promising method to treat CML. For instance, Sp1
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functions as a positive regulator of the BCR-ABL1 gene, and suppression of Sp1 leads to a decrease in BCR-ABL1 expression, which inhibits cell proliferation and the
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Bcr-Abl kinase signaling pathway [30]. In the case of energy restriction, O2 shortage
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reduces BCR-ABL1 mRNA levels and BCR-ABL1 promoter activity by decreasing the level of H4 acetylated at the BCR promoter [31]. Our results suggest that MXD1 functions as a transcriptional repressor that binds to the BCR-ABL1 promoter and
resistance.
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inhibits its expression, giving rise to reductions in CML cell proliferation and TKI
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Combination therapy is necessary for cancer because tumors are genetically
diverse and because drug resistance seems inevitable [32]. BCR-ABL1 overexpression reportedly plays an important role in the generation of mutations. Tang et al reported
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that the kinase domain mutation does not occur in the absence of BCR-ABL1 overexpression [33]. Barnes et al showed that overexpression of Bcr-Abl promotes the appearance of Abl1 kinase domain mutations in imatinib-resistant cell lines [11]. Therefore, downregulation of BCR-ABL1 may also decrease the probability of BCR-ABL1 mutations, which is a critical factor in TKI resistance.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81770111), the National Key Basic Research Program of China (2014CB745201), the Research Program of Health and Family Planning Commission of Shenzhen Municipality (SZXJ2018063), and Special Support Funds
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of Shenzhen for Introduced High-Level Medical Team.
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Authors’ contributions
Chen Huan performed most of the experiments, analyzed the data and drafted the manuscript. Lou Jin collected and provided the clinical samples. Wang Heng, An Na, and Pan Yuming detected the expression levels of BCR-ABL1 in CML patients. Du
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Xin and Zhang Qiaoxia contributed to the direction of experiments and revision of the
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manuscript
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transcriptional control of the MYC oncogene, Mol Cancer. 14 (2015) 132. [21] M. Marega, R.G. Piazza, A. Pirola, S. Redaelli, A. Mogavero, I. Iacobucci, I. Meneghetti, M. Parma, E.M. Pogliani, C. Gambacorti-Passerini, BCR and BCR-ABL regulation during myeloid differentiation in healthy donors and in chronic phase/blast crisis CML patients, Leukemia. 24 (2010) 1445-1449. [22] N.P. Shah, O.N. Witte, C.T. Denny, Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines, Mol Cell Biol. 11 (1991) 1854-1860. [23] Q.S. Zhu, N. Heisterkamp, J. Groffen, Unique organization of the human BCR gene promoter, Nucleic Acids Res. 18 (1990) 7119-7125. [24] M. Conacci-Sorrell, L. McFerrin, R.N. Eisenman, An overview of MYC and its interactome, Cold Spring Harb Perspect Med. 4 (2014) a014357. [25] T.A. Baudino, J.L. Cleveland, The Max Network Gone Mad, Molecular and Cellular Biology. 21 (2001) 691-702. [26] C. Bouchard, O. Dittrich, A. Kiermaier, K. Dohmann, A. Menkel, M. Eilers, B. Lüscher, Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter, Genes Dev. 15 (2001) 2042-2047. [27] E. Jabbour, D. Jones, H.M. Kantarjian, S. O'Brien, C. Tam, C. Koller, J.A. Burger, G. Borthakur, W.G. Wierda, J. Cortes, Long-term outcome of patients with chronic myeloid leukemia treated with second-generation tyrosine kinase inhibitors after imatinib failure is predicted by the in vitro sensitivity of BCR-ABL kinase domain mutations, Blood. 114 (2009) 2037-2043. [28] A. Quintas-Cardama, H. Kantarjian, J. Cortes, Imatinib and beyond--exploring the full potential of targeted therapy for CML, Nat Rev Clin Oncol. 6 (2009) 535-543. [29] K. Keeshan, K.I. Mills, T.G. Cotter, S.L. McKenna, Elevated Bcr-Abl expression levels are sufficient for a haematopoietic cell line to acquire a drug-resistant phenotype, Leukemia. 15 (2001) 1823-1833. [30] X. Yang, J. Pang, N. Shen, F. Yan, L.C. Wu, A. Al-Kali, M.R. Litzow, Y. Peng, R.J. Lee, S. Liu, Liposomal bortezomib is active against chronic myeloid leukemia, Oncotarget. 7 (2016) 36382-36394. [31] S. Bono, M. Lulli, V.G. D'Agostino, F. Di Gesualdo, R. Loffredo, M.G. Cipolleschi, A. Provenzani, E. Rovida, P. Dello Sbarba, Different BCR/Abl protein suppression patterns as a converging trait of chronic myeloid leukemia cell adaptation to energy restriction, Oncotarget. 7 (2016) 84810-84825. [32] A.D. Levinson, Cancer therapy reform, Science. 328 (2010) 137. [33] C. Tang, L. Schafranek, D.B. Watkins, W.T. Parker, S. Moore, J.A. Prime, D.L. White, T.P. Hughes, Tyrosine kinase inhibitor resistance in chronic myeloid leukemia cell lines: investigating resistance pathways, Leuk Lymphoma. 52 (2011) 2139-2147.
Fig. 1. Expression of MXD1 in CML patients and CML cell lines. (A) Differential expression of MXD1 mRNA in the peripheral blood cells of CML patients (n = 91) and healthy donors (n = 55). (B) Relative expression levels of MXD1 and BCR-ABL1 were analyzed with Spearman’s correlation test. (C and D) qRT-PCR and Western blot were used to assess the expression of MXD1 in K562 and K562/G01 cells. (E) Blots were quantified by densitometry relative to the internal control Actin. *P < 0.05; **P
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< 0.01; ***P < 0.001.
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Fig. 2. Effect of MXD1 overexpression on the proliferation and drug resistance of K562 and K562/G01 cells. (A and B) qRT-PCR and Western blot experiments
validated MXD1 overexpression at both the mRNA and protein levels. (C) Blots were
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quantified by densitometry relative to the internal control Actin. (D) CCK assays were performed to measure K562 cell proliferation. (E and F) After treatment of K562 cells
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with imatinib for 48 h, IC50 values were determined by CCK assay.
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(G) Western blotting confirmed the overexpression of MXD1 in K562/G01 cells. (H)
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Blots were quantified by densitometry relative to the internal control Actin. (I and J) CCK assays revealed the IC50 values of K562/G01 cells treated with imatinib for 48 h.
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**P < 0.01; ***P < 0.001.
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Fig. 3. Knockdown of MXD1 and its effect on the proliferation and drug resistance of K562 cells. (A and B) qRT-PCR and Western blot were performed to validate the
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efficiency of MXD1 knockdown. (C) Blots were quantified by densitometry relative to the internal control Actin. (D) CCK assays were used to measure cell proliferation after MXD1 knockdown. (E and F) After treatment of K562 cells with imatinib for 48
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h, the IC50 values were determined by CCK assay. *P < 0.05; **P < 0.01; ***P < 0.001. Fig. 4. Overexpression of MXD1 reduces BCR-ABL1 expression. (A and B) qRT-PCR was applied to determine the BCR-ABL1 mRNA levels in K562 and K562/G01 cells that overexpressed MXD1. Data represent three independent experiments. (C) K562 and K562/G01 cells were infected with lentiviruses overexpressing MXD1 or the
negative control, and Western blot analysis of total cell lysates is shown. Actin was used as the internal control. (D and E) Blots were quantified by densitometry relative to the internal control Actin. *P < 0.05; ***P < 0.001. Fig. 5. MXD1 negatively regulates BCR-ABL1. (A) Dual luciferase reporter assays were performed 48 h after transfection; pGL3-Empty was the negative control. (B)
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Both region1 and region2 were responsible for the MXD1-induced activity of the BCR-ABL1 promoter. Data are presented as relative luciferase values after
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normalization to Renilla luciferase. **P < 0.01; ***P < 0.001.
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S0145-2126(18)30446-6 https://doi.org/10.1016/j.leukres.2018.10.012 LR 6062
To appear in:
Leukemia Research
Received date: Revised date: Accepted date:
27-6-2018 8-10-2018 24-10-2018
Please cite this article as: Huan C, Jin L, Heng W, Na A, Yuming P, Xin D, Qiaoxia Z, MXD1 regulates the imatinib resistance of chronic myeloid leukemia cells by repressing BCR-ABL1 expression, Leukemia Research (2018), https://doi.org/10.1016/j.leukres.2018.10.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page Title: MXD1 regulates the imatinib resistance of chronic myeloid leukemia cells by repressing BCR-ABL1 expression Author names: Chen Huan, Lou Jin, Wang Heng, An Na, Pan Yuming, Du Xin *, and Zhang Qiaoxia * Author affiliations: Shenzhen Bone Marrow Transplantation Public Service Platform,
Affiliated Hospital of Shenzhen University, Shenzhen, China
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Shenzhen Institute of Hematology, Shenzhen Second People's Hospital, The First
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Corresponding author: Zhang Qiaoxia, [email protected], 13925263107 Present address: NO. 3002 Sungang West Road, Futian District, Shenzhen City, Guangdong Province, China
Transcriptional repressor MXD1 shows low expression in CML patients and imatinib-resistant K562/G01 cell line
Overexpression of MXD1 inhibits the proliferation of K562 cells and sensitize
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K562/G01 cell line to imatinib
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Highlights
MXD1 downregulates BCR-ABL1 expression by inhibiting the transcriptional
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Abstract
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activity of the BCR-ABL1 gene promoter.
Tyrosine kinase inhibitors have achieved unprecedented efficacy in the treatment
of chronic myeloid leukemia (CML); however, imatinib resistance has emerged as a
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major problem in the clinic. Because the overexpression of BCR-ABL1 critically contributes to CML pathogenesis and drug resistance, targeting the regulation of BCR-ABL1 gene expression may be an alternative therapeutic strategy. In this study, we found that the transcriptional repressor MXD1 showed low expression in CML patients and was negatively correlated with BCR-ABL1. Overexpression of MXD1 markedly inhibited the proliferation of K562 cells and sensitized the imatinib-resistant
K562/G01 cell line to imatinib, with decreased BCR-ABL1 mRNA and protein expression. Further investigation using reporter gene analysis showed that MXD1 significantly inhibited the transcriptional activity of the BCR-ABL1 gene promoter. Taken together, these data show that MXD1 functions as a negative regulator of BCR-ABL1 expression and subsequently inhibits proliferation and sensitizes CML
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cells to imatinib treatment.
Keywords: Chronic myeloid leukemia, MXD1, BCR-ABL1, Tyrosine kinase inhibitors,
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Drug resistance
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1. Introduction
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Chronic myeloid leukemia (CML) is a common myeloproliferative neoplasm
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characterized by the chromosomal translocation t(9;22) (q34;q11) [1]. This translocation causes the generation of a fusion oncogene, namely, the BCR-ABL1
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encoding fusion protein with constitutive tyrosine kinase activity, which gives rise to
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the uncontrolled growth of myeloid cells in the bone marrow through a series of downstream pathways [2,3]. One of the first-line treatments for CML is imatinib, a tyrosine kinase inhibitor (TKI) that competitively binds to the ATP-binding site of
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Bcr-Abl and blocks the downstream signal pathway [4,5]. TKIs have dramatically improved the overall survival rate of CML patients [6,7]. However, TKI resistance has
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become a major clinical problem for CML, mainly as a consequence of BCR-ABL1 mutations, BCR-ABL1 overexpression and other BCR-ABL1 independent pathways [8,9]. Bcr-Abl protein abundance heavily contributes to CML pathogenesis and drug
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resistance. Additionally, the expression level of BCR-ABL1 significantly influences the generation of BCR-ABL1 mutations [10,11]. One suggestion is that targeting Bcr-Abl fusion protein expression might override aberrant Abl kinase activity. However, the molecular mechanism of BCR-ABL1 gene regulation needs to be further characterized. MAX dimerization protein 1 (MXD1) is a transcription factor that belongs to the
MYC/MXD/MAX
family,
whose
members
typically
contain
a
basic
helix-loop-helix-leucine-zipper (bHLHZip) domain [12]. With this domain, MXD1 binds to the E-box DNA element in the promoters of target genes to regulate their transcription. As a transcriptional repressor, MXD1 mainly influences cell transformation, proliferation, apoptosis, and differentiation [13,14,15]. MXD1 has been reported to be an antagonist of MYC that inhibits cell proliferation and
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antagonizes the transforming activity of MYC [16]. MXD1 is also involved in the survival, migration and invasion of cancer cells, such as pancreatic cancer cells,
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gastric cancer cells and lung cancer cells [17,18,19]. To date, the role of MXD1 in CML has not been clarified. Since MXD1 E-box consensus binding sites are present in
the promoter of BCR-ABL1 [20], we aimed to determine whether MXD1 regulates the
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expression of BCR-ABL1.
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In this study, we investigated the expression and biological function of MXD1 in
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CML patients, K562 cells and imatinib-resistant K562/G01 cells. The results showed that the level of MXD1 was significantly lower in CML patients and K562/G01 cells
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than in controls. In addition, MXD1 inhibited the proliferation and enhanced the
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imatinib sensitivity of K562 cells and K562/G01 cells by reducing BCR-ABL1 gene expression.
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2. Materials and Methods
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2.1. Cell lines and human samples Human CML cell lines K562 and K562/G01 (imatinib induced resistance) were
purchased from the Shanghai Institute of Life Sciences (Shanghai, China) and State
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Key Laboratory of Experimental Hematology (Tianjing, China), respectively. Cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37˚C in 5% CO2. To maintain drug resistance, imatinib (1 µg/ml) was applied to K562/G01 cells at least 2 weeks before experiments. The peripheral blood cells of CML patients and healthy donors were collected at Shenzhen Second People’s Hospital. In total, 91 cases of CML patients and 55 cases
of normal control were enrolled. All CML patients had clinical data on their BCR-ABL1 (p210) levels, shown as the percentage of p210 to ABL as measured by qRT-PCR. All participants signed informed consent forms, and all studies involving the use of human blood were performed in accordance with the Declaration of Helsinki. This study was approved by the Ethics Committee of Shenzhen Second
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People’s Hospital.
2.2. RNA isolation and quantitative real-time PCR analysis
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Total RNA was extracted from cultured cells with RNAiso Plus (Takara, JAP). Reverse transcription into cDNA was carried out with the PrimeScriptTM RT reagent kit (Takara, JAP) according to the manufacturer’s protocol. Real-time PCR was
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performed with SYBR® Premix Ex Taq™ II (Takara, JAP). The relative expression
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level was determined with the 2-∆∆Ct method, and the human GAPDH gene was
ATTCGGGTCCAAGTG-3’
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5’-TGAACATGGTTATGCCTCCA-3’
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applied as the internal control. The primer sequences were as follows: MXD1: (forward),
5’-ACTTG
(reverse);
BCR-ABL1:
5’-TCCACTGGCCACAAAATCATACAG-3’
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5’-TCAGAAGCTTCTCCCTGACATCCG-3’ GTCAAGGCTGAGAACGGGAA-3’
(forward), (reverse);
and
(forward),
GAPDH:
5’5’-
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TCGCCCCACTTGATTTTGGA-3’ (reverse). Thermal cycling reactions were performed as follows: 95˚C for 5 min, followed by 40 cycles of two-step PCR at 95˚C
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for 5 sec and 60˚C for 34 sec.
2.3 Establishment of stably transfected cell lines
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MXD1-overexpressing lentivirus vector (pHBLV-MXD1-ZsGreen-Puro, flag) and control lentivirus vector (pHBLV-ZsGreen-Puro) were constructed and then enveloped to produce lentiviruses. After target cell infections, puromycin selection was applied to establish the stably transfected K562 cell lines. To produce lentiviruses, 293T packaging cells were cotransfected with lentivirus and packaging plasmids, namely, pSPAX2 and pMD2G, respectively. Before transfection, 293T cells were
plated in a 10-cm dish and cultured in DMEM (Gibco, USA) with 10% FBS (Gibco, USA). When the cell density reached 60~80%, 293T cells were transfected with the plasmids mentioned above. 12~16 h later, the culture medium was replaced with 5~6 ml of fresh DMEM with 10% FBS. Then, the supernatants, which contained virus, were harvested at 24 h and 48 h and concentrated by ultracentrifugation for 90 min at 50,000 g and 4°C. knock
down
MXD1,
shRNA
oligonucleotides
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(GGACCCGAATCAAGTCGACACACTA) were designed using ABI online software, the
corresponding
sequence
TTCTCCGAACGTGTCACGTAA.
of
scrambled
shRNA
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and
RNA
interference
was
vector
(pHBLV-U6-MXD1-Puro) and control lentivirus were constructed. After target cell
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infections, puromycin was added to screen positive cells with stably reduced
2.4 Cell proliferation and viability assays
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expression of MXD1.
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To test cell proliferation, a Cell Counting Kit (CCK, TransGen Biotech, China)
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was used. K562 cells were seeded into 96-well plates. Every 24 h, CCK reagents were added to each well in a 10 µl volume, the plates were incubated at 37˚C for 2 h, and the absorbance was measured at 450 nm.
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The CCK kit was also used for a cell viability assay. K562 or K562/G01 cells were seeded into 96-well plates and treated with imatinib to determine IC50 values.
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K562 cells were treated with a series of imatinib concentrations (0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 and 5.12 µM), and K562/G01 cells were treated with a series of imatinib concentrations (0, 0.5, 1, 2, 4, 8, 16, 32 and 64 µM). After 48 h, CCK
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reagents were added to each well in a volume of 10 µl, the plates were incubated at 37˚C for 2 h, and the absorbance was measured at 450 nm.
2.5 Western blot Total protein was extracted using RIPA lysis buffer (Thermos Fisher, USA) supplemented with a protease inhibitor cocktail tablet (Roche Applied Science). The
protein content was measured with a BCA protein assay kit (Thermo Fisher, USA). Protein samples (20 mg/lane) were resolved on a 10% bis-tris gel (SDS-PAGE) and transferred onto 0.22-µm PVDF membranes (Millipore, USA). After blotting, the membranes were blocked with 3% BSA in TBS-Tween for 2 h and incubated with relevant primary antibodies overnight at 4˚C. These antibodies included anti-Mxd1 (Proteintech, USA, 17888-1-AP), anti-c Abl (Cell Signaling Technology, USA,
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#2862s) and anti-Actin (Abcam, UK, ab3280). The membranes were washed three times with 1x TBS supplemented with 0.1% Tween 20, hybridized with HRP-labeled
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secondary antibodies—anti-rabbit IgG (Cell Signaling Technology, USA, #7074) and
anti-mouse IgG (Cell Signaling Technology, USA, #7076) and then visualized with a
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chemiluminescence detection reagent (Thermos Fisher, USA).
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2.6 Dual luciferase reporter assay
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pGL3-BCR promoter constructs are referred to in a previous report by Marega et al [21]. A sequence 1455 bp upstream from the ATG site of the BCR gene and several
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truncated sequences were synthesized by BGI (China). After digestion with Kpn1 and
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Xhol (Roche, USA), these sequences were inserted into the pGL3 vectors separately. 293T cells were cotransfected with pCMV3-MXD1 (Sinobiological, China) expression vectors and pGL3-BCR luciferase reporter vectors, along with pRL-TK
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(Promega, USA) vectors, by using Lipofectamine 3000 (Invitrogen, USA). Forty-eight hours after transfection, cells were harvested, and a Dual Luciferase
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Reporter Assay System (Promega, USA) was used to determine the luciferase activities according to the manufacturer’s instructions. Relative luciferase activity was
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normalized to the Renilla luciferase signal.
2.7 Statistical analysis All experiments were repeated at least three times, and data are presented as the mean ± SD. The differences were calculated using the unpaired t-test, and Spearman’s correlation was performed with GraphPad Prism 5 software. P < 0.05 was deemed statistically significant.
Results
3.1 MXD1 is downregulated in CML patients and K562/G01 cells We compared the relative levels of MXD1 mRNA in the peripheral blood cells of CML patients and healthy donors by qRT-PCR. The results showed that the MXD1
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mRNA level in CML patients was significantly lower than that in the healthy donors (0.62 ± 0.05 vs 1.25± 0.11, respectively; P < 0.001) (Fig. 1A). A further correlation
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analysis indicated that the mRNA levels of BCR-ABL1 and MXD1 were negatively correlated in CML patients (Fig. 1B). We also detected the expression of MXD1 in wild type CML cell line K562 and imatinib-resistant K562/G01 cells. The results
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showed that K562/G01 cells expressed less MXD1 mRNA and protein than K562 cells
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(Fig. 1C and D).
3.2 Overexpression of MXD1 reduces the proliferation and imatinib resistance of
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CML cells
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As the expression of MXD1 was downregulated in the CML cells, to further examine the roles of MXD1 in CML, we established a stably overexpressing MXD1 K562 cell line using recombinant lentivirus. As expected, the expression of MXD1
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was much higher in the MXD1-overexpression K562 cells (K562-MXD1) than in the control cells (K562-Con, K562 cells stably transfected with control lentivirus), as
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shown by qPCR and Western blot analyses (Fig. 2A and B). The results of the CCK assays showed that overexpression of MXD1 inhibits K562 cell proliferation (Fig. 2D). To determine the effect of MXD1 on the imatinib sensitivity of K562 cells, stably
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transfected K562 cells were treated with a series of concentrations of imatinib (0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 and 5.12 µM) for 48 h, and then, the IC50 value was calculated. The result showed that MXD1 significantly decreased the IC50 value from (0.44 ± 0.01 µM) to (0.21 ± 0.01 µM) (P < 0.001), suggesting that overexpression of MXD1 enhanced the sensitivity of K562 cells to imatinib treatment (Fig. 2E and F). To further verify the effect of MXD1 on the TKI sensitivity of CML cells, an
imatinib-resistant cell line, namely, K562/G01, was used as a study model. We added lentivirus, including MXD1 cDNA or control lentivirus, to the K562/G01 cells and used puromycin to select stably transfected cells, that is, K56/G01-MXD1 and K562/G01-Con cells. Then, the K562/G01-MXD1 cells were treated with a series of concentrations of imatinib (0, 0.5, 1, 2, 4, 8, 16, 32 and 64 µM). Overexpression of
relative to K562/G01-Con cells (4.09 ± 0.18 µM), P < 0.01 (Fig. 2G-J).
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MXD1 drastically reduced the IC50 value of K562/G01-MXD1 cells (8.20 ± 0.51 µM)
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3.3 Knockdown of MXD1 increases the proliferation and drug resistance of K562 cells
K562 cells were stably transfected with lentivirus encoding shMXD1 and
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scrambled shRNA (negative control: shNC). The shMXD1 silencing efficiency was
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determined by qRT-PCR and Western blot; MXD1 mRNA and protein levels were
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obviously decreased in K562-shMXD1 cells (79.98% and 35.58%, respectively) relative to K562-shNC cells (Fig. 2A and C). CCK assays showed that knockdown of
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MXD1 promoted the proliferation of K562 cells (Fig. 3D). However, after treatment
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with a series of concentrations of imatinib (0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 and 5.12 µM) for 48 h, the IC50 value of K562-shMXD1 cells (0.78 ± 0.05 µM) was increased significantly relative to that of K562-shNC cells (0.33 ± 0.02 µM), which
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indicated that downregulation of MXD1 increased the imatinib resistance of K562
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cells (Fig. 3E and F).
3.4 MXD1 downregulates BCR-ABL1 expression by transcriptional inhibition We have shown that BCR-ABL1 and MXD1 are negatively correlated in CML
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patients (Fig. 1B). Similarly, BCR-ABL1 mRNA and protein levels are downregulated by overexpressed MXD1 in K562 cells and in K562/G01 cells relative to negative controls (Fig. 4A-C). These results suggest that MXD1 is involved in BCR-ABL1 expression. To determine the mechanism by which MXD1 regulates BCR-ABL1, we cloned
BCR promoter constructs, with a sequence starting 1455 bp upstream of the transcriptional start site of the BCR gene, into a luciferase reporter vector and named the vector pGL3-BCR1, and 293T cells were used to conduct luciferase reporter assays. The results indicated that overexpression of MXD1 dramatically decreased the luciferase activity of pGL3-BCR1 but not pGL3-Empty (negative control), suggesting that MXD1 significantly repressed the promoter activity of BCR (Fig. 5A).
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According to Marega et al [21] and previous studies [22,23], there are two
potential regions for the transcriptional regulation of MXD1 in the BCR gene, namely,
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region1, from -1425 bp to -1196 bp, and region2, from -883 bp to -690 bp. Thus, we constructed another three vectors, as follows: pGL3-BCR2: 1455 bp upstream of the BCR coding starting site with deletion of region1; pGL3-BCR3: 1455 bp upstream of
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the BCR coding starting site with deletion of region2; pGL3-BCR4: 1455 bp upstream
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of the BCR coding starting site with deletion of both region1 and region2. The results
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showed that in the presence of MXD1, both pGL3-BCR2 and pGL3-BCR3 had reduced luciferase activity (P < 0.001). However, deletions of both region1 and
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region2 almost completely abrogated the regulation of BCR promoter activity by
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MXD1, suggesting the important roles of these two regions in the transcription of BCR
Discussion
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(Fig. 5B).
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MXD1 is an important transcriptional repressor, and the bHLHZip domain
enables MXD1 binding to a specific DNA site, namely, the CACGTG E-box element [24]. Another key structural element of MXD1 is the mSIN3 interaction domain (SID),
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which is able to recruit a mSIN3-HDAC repressor complex and then acetylates core histones [25]. Lee et al [14] demonstrated that MXD1 is a transcriptional repressor of Bcl-6, which is a major regulator of B cell neoplastic transformation, and MXD1 directly regulates Bcl-6 transcription via the E-box-like DNA sequences in the Bcl-6 upstream promoter region. Bouchard et al [26] found that in differentiating HL60 cells, MXD1/MAX complexes bind to the cyclin D2 promoter in vivo and increase
histone deacetylation, thereby regulating cyclin D2 gene expression. In summary, MXD1 can regulate the compaction and accessibility of chromatin and control the expression of different genes. Our results showed that MXD1 downregulated BCR-ABL1 expression at the mRNA and protein levels and inhibited the proliferation and increased the TKI sensitivity of CML cells.
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Although TKI imatinib has shown remarkable effects on CML therapy and improved the overall survival of patients, drug resistance and relapse tend to develop
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after long-term application [27,28]. As BCR-ABL1 levels play a critical role in its tyrosine kinase activity, drug resistance, the formation of point mutations and disease progression [8,10,29], targeting the expression of BCR-ABL1 might be an alternative
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strategy for CML treatment. Recently, several studies have shown that decreasing
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BCR-ABL1 expression represents a promising method to treat CML. For instance, Sp1
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functions as a positive regulator of the BCR-ABL1 gene, and suppression of Sp1 leads to a decrease in BCR-ABL1 expression, which inhibits cell proliferation and the
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Bcr-Abl kinase signaling pathway [30]. In the case of energy restriction, O2 shortage
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reduces BCR-ABL1 mRNA levels and BCR-ABL1 promoter activity by decreasing the level of H4 acetylated at the BCR promoter [31]. Our results suggest that MXD1 functions as a transcriptional repressor that binds to the BCR-ABL1 promoter and
resistance.
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inhibits its expression, giving rise to reductions in CML cell proliferation and TKI
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Combination therapy is necessary for cancer because tumors are genetically
diverse and because drug resistance seems inevitable [32]. BCR-ABL1 overexpression reportedly plays an important role in the generation of mutations. Tang et al reported
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that the kinase domain mutation does not occur in the absence of BCR-ABL1 overexpression [33]. Barnes et al showed that overexpression of Bcr-Abl promotes the appearance of Abl1 kinase domain mutations in imatinib-resistant cell lines [11]. Therefore, downregulation of BCR-ABL1 may also decrease the probability of BCR-ABL1 mutations, which is a critical factor in TKI resistance.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81770111), the National Key Basic Research Program of China (2014CB745201), the Research Program of Health and Family Planning Commission of Shenzhen Municipality (SZXJ2018063), and Special Support Funds
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of Shenzhen for Introduced High-Level Medical Team.
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Authors’ contributions
Chen Huan performed most of the experiments, analyzed the data and drafted the manuscript. Lou Jin collected and provided the clinical samples. Wang Heng, An Na, and Pan Yuming detected the expression levels of BCR-ABL1 in CML patients. Du
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Xin and Zhang Qiaoxia contributed to the direction of experiments and revision of the
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manuscript
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Fig. 1. Expression of MXD1 in CML patients and CML cell lines. (A) Differential expression of MXD1 mRNA in the peripheral blood cells of CML patients (n = 91) and healthy donors (n = 55). (B) Relative expression levels of MXD1 and BCR-ABL1 were analyzed with Spearman’s correlation test. (C and D) qRT-PCR and Western blot were used to assess the expression of MXD1 in K562 and K562/G01 cells. (E) Blots were quantified by densitometry relative to the internal control Actin. *P < 0.05; **P
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< 0.01; ***P < 0.001.
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Fig. 2. Effect of MXD1 overexpression on the proliferation and drug resistance of K562 and K562/G01 cells. (A and B) qRT-PCR and Western blot experiments
validated MXD1 overexpression at both the mRNA and protein levels. (C) Blots were
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quantified by densitometry relative to the internal control Actin. (D) CCK assays were performed to measure K562 cell proliferation. (E and F) After treatment of K562 cells
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with imatinib for 48 h, IC50 values were determined by CCK assay.
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(G) Western blotting confirmed the overexpression of MXD1 in K562/G01 cells. (H)
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Blots were quantified by densitometry relative to the internal control Actin. (I and J) CCK assays revealed the IC50 values of K562/G01 cells treated with imatinib for 48 h.
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Fig. 3. Knockdown of MXD1 and its effect on the proliferation and drug resistance of K562 cells. (A and B) qRT-PCR and Western blot were performed to validate the
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efficiency of MXD1 knockdown. (C) Blots were quantified by densitometry relative to the internal control Actin. (D) CCK assays were used to measure cell proliferation after MXD1 knockdown. (E and F) After treatment of K562 cells with imatinib for 48
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h, the IC50 values were determined by CCK assay. *P < 0.05; **P < 0.01; ***P < 0.001. Fig. 4. Overexpression of MXD1 reduces BCR-ABL1 expression. (A and B) qRT-PCR was applied to determine the BCR-ABL1 mRNA levels in K562 and K562/G01 cells that overexpressed MXD1. Data represent three independent experiments. (C) K562 and K562/G01 cells were infected with lentiviruses overexpressing MXD1 or the
negative control, and Western blot analysis of total cell lysates is shown. Actin was used as the internal control. (D and E) Blots were quantified by densitometry relative to the internal control Actin. *P < 0.05; ***P < 0.001. Fig. 5. MXD1 negatively regulates BCR-ABL1. (A) Dual luciferase reporter assays were performed 48 h after transfection; pGL3-Empty was the negative control. (B)
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Both region1 and region2 were responsible for the MXD1-induced activity of the BCR-ABL1 promoter. Data are presented as relative luciferase values after
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normalization to Renilla luciferase. **P < 0.01; ***P < 0.001.
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