Inhibition of c-Myc Overcomes Cytotoxic Drug Resistance in Acute Myeloid Leukemia Cells by Promoting Differentiation

Nowadays, drug resistance still represents a major obstacle to successful acute myeloid leukemia (AML) treatment and the underlying mechanism is not fully elucidated. Here, we found that high expression of c-Myc was one of the cytogenetic characteristics in the drug-resistant leukemic cells. c-Myc over-expression in leukemic cells induced resistance to chemotherapeutic drugs, enhanced colony formation capacity and inhibited cell differentiation induced by all-trans retinoic acid (ATRA). Meanwhile, inhibition of c-Myc by shRNA or specific c-Myc inhibitor 10058-F4 rescued the sensitivity to cytotoxic drugs, restrained the colony formation ability and promoted differentiation. RT-PCR and western blotting analysis showed that down-regulation of C/EBPβ contributed to the poor differentiation state of leukemic cells induced by c-Myc over-expression. Importantly, over-expression of C/EBPβ could reverse c-Myc induced drug resistance. In primary AML cells, the c-Myc expression was negatively correlated with C/EBPβ. 10058-F4, displayed anti-proliferative activity and increased cellular differentiation with up-regulation of C/EBPβ in primary AML cells. Thus, our study indicated that c-Myc could be a novel target to overcome drug resistance, providing a new approach in AML therapy.


Introduction
Acute myeloid leukemia (AML) is a kind of aberrant clonal hematopoietic malignancy originating from genetic mutations, or epigenetic aberrations in normal hematopoietic progenitors. Although chemotherapy results in clinical remission for AML, drug resistance still represents a huge challenge to achieve effective treatment, especially in relapsed or refractory AML patients [1,2]. Cytostatic drug resistance occurs through different mechanisms, including induction of drug detoxification [3], the over-expression of oncogene or inactivation of tumor suppressor gene [4,5], metabolic disturbance [6,7], as well as existence of leukemia stem cells [8]. Still, the exact mechanisms of drug resistance in leukemia have not been fully investigated. Elucidation of the intrinsic or acquired factors that mediate drug resistance remains of critical importance for the development of novel therapeutic strategies for relapsed or refractory AML patients.
The proto-oncogene c-Myc plays a pivotal role in cellular metabolism [9], apoptosis [10], differentiation [11], cell cycle progress [12], and tumorigenesis [13]. c-Myc encodes a basic helixloop-helix leucine zipper transcription factor, which transcripts an array of downstream target genes [14]. c-Myc, as an attractive target for cancer therapy, is aberrantly expressed in a wide variety of human solid tumors [15] as well as leukemia [16]. Study reported that c-Myc over-expression was closely correlated to chemotherapy resistance in salivery carcinoma [17]. Inhibition of c-Myc overcame drug resistance in some cancers, such as Lewis lung carcinoma [18] and melanoma [19]. c-Myc antisense oligodeoxynucleotides increased cisplatin sensitivity in metastatic melanoma cell lines inherently resistant to cisplatin [20]. 10058-F4, a targeted inhibitor of c-Myc, was reported to be effective in anti-tumor treatment, such as hepatocellular carcinoma [21] and leukemia [22]. However, the precise role of c-Myc in drug resistance of leukemic cells has not yet been elucidated.
In this study, we identified the effects of c-Myc on drug resistance in leukemic cell lines and AML primary cells. We found that the up-regulated expression of c-Myc in leukemic cells promoted colony formation ability and maintained poor differentiation mediated by suppression of C/EBPb, leading to drug resistance. Consistently, down-regulation of c-Myc abrogated colony formation capacity of leukemic cells and promoted cellular differentiation. Our study provided a new approach to overcome drug resistance by c-Myc inhibition in AML therapy.

Cell cytotoxicity Assay
Cell cytotoxicity was evaluated by MTT assay according to the manufacture's instruction. Briefly, leukemic cells or primary BMMCs were seeded in the 96-well U-bottomed plate. Subsequently, cells were treated with different drugs at different concentrations for indicated time. After MTT solution was added to each well, cells were incubated at 37uC for another 4 h and the absorbance was finally determined at 490 nm using the microplate reader (BioTek, Vermont, USA).

Colony formation assay
Cells with different drugs treatment were plated in 10% FBS medium with methylcellulose (R&D Systems, Minneapolis, MN, USA) for 6 days. The colony forming units (CFU) were counted using microscopy (IX81, Olympus, Japan), and the colony number was the sum of randomized forty visions.

Retroviral and lentiviral transduction
The MSCV 2.2 vector was kindly provided by Prof. Hua Huang (University of Colorado, Denver, USA). The c-Myc and C/EBPb gene were cloned and inserted into the MSCV 2.2 vector. Retrovirus was produced from packaging cell line GP2-293 with the aid of pseudo-envelope vector. Lentiviral vector expressing short hairpins against human c-Myc (ShMYC) was constructed within the lentivirus plasmid vector pLL3.7 using the target sequence 59-CCATAATGTAAACTGCCTCAA-39. Lentiviral particles were produced by cotransfection of 293T cells with packaging vectors (pMD2.G and pPAX2). The virus supernatants were collected 48 h after transfection.

Cell morphology analysis
Cells were seeded into 6-well plates and incubated with specific drug for indicated time and collected for analysis. Cytospin slides were prepared, and the cells were counterstained with Wright-Giemsa. The morphology of cells was observed by microscopy (7641, Olympus, Japan).

Western blotting analysis
Total cellular proteins were isolated with lysis buffer. Equal amounts of protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked and then incubated with c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), C/EBPb (Abcam, Cambridge, UK) and GAPDH antibody (Cell Signaling Technology Corp, Beverly, MA, USA). The protein bands were visualized using an enhanced chemiluminescence reagent (Sigma, St. Louis, MO, USA), according to the manufacturer's instruction.

Flow cytometry analysis
Analysis of CD11b expression was performed using the PE antihuman CD11b antibody (BD Biosciences, San Jose, CA, USA) for predicting the cellular differentiation.

Nitrotetrazolium blue chloride (NBT) reduction assay
The same amount of cells untreated or treated with ATRA for 72 h were incubated at 37uC for 1 h in RPMI 1640 medium containing 10% FBS and 0.1% NBT (Sigma-Aldrich, MO, USA). The cells were then centrifuged and dissolved in DMSO. The optical density was read at 570 nm.

RNA extraction and PCR analysis
RNA was isolated using Trizol reagent (Invitrogen, Grand Island, NY, USA), and reverse transcribed using M-MuLV reverse transcriptase (Fermentas, St Leon-Roth, Germany). Real-time PCR was performed using SYBR Green qPCR mix (Toyabo, Osaka, Japan) on Applied Biosystems 7500 Fast Real-Time PCR thermocycler (Applied Biosystems, Foster City, CA, USA) and quantified using ABI 7500 software.

Statistical analysis
Data were presented as mean 6 SD. Statistical analysis was performed using SPSS v. 16.0 (SPSS, Inc.) and GraphPad Prism 6 (GraphPad Software, Inc.). A value of p,0.05 was considered statistically.

c-Myc is high expressed in drug resistant leukemic cells
To find the cytogenetic characteristics in drug-resistant leukemic cells, two drug-resistant leukemic cell lines were applied for detecting the properties. NB4-R2 and K652/G were respectively resistant to all-trans-retinoic acid (ATRA) and imatinib, compared with their parental cell lines (NB4 and K562), as shown in Figure  S1. The anti-proliferative effect of cytarabine (Ara-C), daunomycin (DNR) or doxorubicin (Doxo), clinical cytotoxic drugs for treating AML, was detected by MTT assay ( Figure 1A). The IC50 values of Ara-C, DNR or Doxo at 48 h were 0.3900 mM, 0.2097 mg/ml or 0.0469 mg/ml respectively for NB4, 1.4770 mM, 0.4540 mg/ml or 0.0838 mg/ml for NB4-R2, 0.8010 mM, 0.3325 mg/ml or 0.0827 mg/ml for K562, and 6.0368 mM, 0.7375 mg/ml or 0.2063 mg/ml for K562/G. The result showed that NB4-R2 and K652/G were both resistant to Ara-C, DNR or Doxo, indicating that the two resistant leukemic cells had a broadspectrum of resistance to clinical cytotoxic drugs. The colony number of NB4-R2 cells was more than that of NB4 cells under 6 days' culture ( Figure 1B-a). Similar phenomenon was observed in K562/G cells compared to K562 cells ( Figure 1B-b). The data indicated drug-resistant cell lines also displayed increased colony formation capacity. Chemotherapy resistance was closely correlated to c-Myc amplification, we thus testify whether c-Myc over-expression was involved in the drug-resistant leukemic cells. Western blotting assay revealed that the expression level of c-Myc was higher in drug-resistant cells (NB4-R2 and K562/G) than in the control cells ( Figure 1C), suggesting that high c-Myc expression was correlated with enhanced drug-resistance and colony formation capacity in leukemic cells.
c-Myc over-expression contributes to drug resistance and high colony formation capacity in leukemic cells In order to identify the effect of c-Myc on drug resistance and colony formation in leukemia, we constructed the stable leukemic cell line with c-Myc over-expression or knock-down for colony formation assay and drug sensitivity test. The stable leukemic cell line with c-Myc over-expression (NB4/MYC) was derived from the drug-sensitive NB4 cells infected with MSCV retrovirus with c-Myc gene while NB4 cells with MSCV-GFP vector was used as the control (NB4/GFP). Lentiviral vector expressing short hairpins against human c-Myc (ShMYC) was used to reduce c-Myc expression in drug-resistant cell line NB4-R2 (NB4-R2 ShMYC), while lentiviral virus with the non-target ShRNA acted as the control (NB4-R2 Con). Western blotting showed that c-Myc expression was increased in NB4/MYC cells, while decreased in NB4-R2 ShMYC ( Figure 2A). Next, the colony formation assay showed that c-Myc over-expression enhanced the colony formation ability in NB4 cells, and that reduced c-Myc expression restrained the colony formation in drug-resistant cells NB4-R2, as shown in Figure 2B. Drug sensitivity test based on MTT assay was performed to testify the cytotoxic effect with Ara-C, DNR or Doxo treatment when up-or down-regulation of c-Myc in leukemic cells. Cells were treated with Ara-C (0.1 mM), DNR (0.2 mg/ml) or Doxo (0.04 mg/ml) for 48 h. The rate of cell viability of NB4/GFP vs NB4/MYC in treatment with the indicated concentration of Ara-C, DNR or Doxo was 61.38%63.26% vs 70.86%63.75%, 59.82%64.51% vs 68.37%64.13%, or 60.85%64.85% vs 80.64%63.83% respectively, while the rate of NB4-R2 Con vs NB4-R2 ShMYC was 69.57%62.12% vs 47.41%61.74%, 80.89%62.38% vs 67.19%62.14%, or 80.70%62.32% vs 60.21%62.37% respectively, as shown in Figure 2C. Thus, increased c-Myc expression in NB4 cells promoted drug resistance to Ara-C, DNR or Doxo. In contrast, decreased c-Myc expression in drug-resistant cells NB4-R2 rescued the sensitivity to the cytotoxic drugs. Similar phemonemon was also observed in U937 cells ( Figure S2A-2C).
c-Myc inhibits cell differentiation induced by ATRA c-Myc acted as a key transcriptional regulator to maintain stemness in cancer stem like cells [23,24]. c-Myc was also proposed to play a role in the transition from proliferation to differentiation in oligodendrocyte progenitor cells [25]. To explain the mechanism that high c-Myc expression induced drug resistance and promoted colony formation capacity in leukemic cells, we used the cell differentiation model of NB4 cells induced by ATRA. The result showed that the percentage of CD11b-positive cells induced by ATRA was significantly decreased from 35.8%64.0% to 7.4%62.0% after c-Myc over-expression in NB4 cells ( Figure 3A). As well, NB4/GFP cells treated with ATRA represented nucleolus disappearance and obvious nuclear segmentation by Wright-Giemsa staining. However, this phenotype was reversed in NB4/ MYC cells ( Figure 3B). The retard of maturation resulting from c-Myc over-expression was also detected by NBT reduction assay ( Figure 3C). Consistantly, c-Myc over-expression also inhibited U937 cells differentiation induced by ATRA ( Figure S2D-2F). Contrarily, NB4-R2 ShMYC cells with reduced expression of c-Myc showed more CD11b-positive cells after ATRA treatment than the control cells ( Figure 3D). The result was consistent with morphologic observation and NBT reduction assay ( Figure 3E,  3F). Thus, the drug resistant cells were inclined to maintain poorly differentiated state resulting from elevated expression of c-Myc.
Over-expression of C/EBPb reverses c-Myc induced drug resistance C/EBPb was essential in the process of granulocytic differentiation. The DNA binding capacity and protein expression level of C/EBPb were increased evidently in ATRA-induced differentiation of APL cells [26]. Here, we assessed whether high colony formation ability and dedifferentiation state of drug resistant leukemic cells induced by c-Myc was through manipulating C/ EBPb. RT-PCR and western blotting analysis were performed to further validate the expression of C/EBPb. C/EBPb was increased after ATRA treatment in NB4/GFP cells, but high cmyc expression inhibited the up-regulation of C/EBPb in both mRNA and protein levels (35KD and 38KD isoforms) after ATRA treatment ( Figure 4A). The data suggested that C/EBPb was especially down-regulated by c-Myc in ATRA-induced differentiation. To testify the effect of C/EBPb in drug resistance, C/EBPb was over-expressed in NB4/MYC cells (NB4/MYC C/ EBPb) by transient retrovirus transduction (Figure 4B-a). MTT assay was carried out to detect the drug sensitivity of NB4/MYC C/EBPb cells treated with Ara-C (0.1 mM), DNR (0.2 mg/ml) or Doxo (0.04 mg/ml). Result showed that C/EBPb could rescue drug sensitiviy in NB4/MYC cells ( Figure 4B-b, c, d), futher indiating that down-regulation of C/EBPb was involved in drug resistance induced by c-Myc.
c-Myc inhibitor 10058-F4 restrains drug resistance and colony formation ability induced by c-Myc overexpression c-Myc was reported to dimerize with Max to play a part in gene transcription. 10058-F4, a small-molecule c-Myc inhibitor, specifically disrupts the c-Myc/Max heterodimerization, resulting in inhibiting the transcriptional function of c-Myc [27]. To clarify the effect of 10058-F4 on drug-resistant leukemic cells, MTT assay was performed to detect the anti-proliferative effect of 10058-F4. Result showed that the drug-resistant cell lines (NB4-R2 and NB4/MYC) were more sensitive to 10058-F4 than the drugsensitive cell lines (NB4 and NB4/GFP) in both dose-and timedependent manners ( Figure 5A). We also found that the colony formation capacity of both drug-resistant and -sensitive cells were completely impeded by 10058-F4 ( Figure 5B). In addition, 10058-F4 promoted the sensitivity of drug resistant cells responsing to Doxo treatment ( Figure 5C). The data suggested that 10058-F4 could act as a new targeted therapeutic drug to improve the efficacy of chemotherapy in drug resistant leukemic cells.

Inhibition of c-Myc suppresses proliferation and induces differentiation in primary AML cells
To futher investigate the relation between c-Myc and C/EBPb in primary AML cells, BMMCs from AML patients were collected, which contained 5 refractory and relapsed (R) AML patients and 4 newly diagnosed (N) AML patients. RT-PCR analysis showed that the expression of c-Myc was higher in refractory and relapsed patients than in newly diagnosed patients ( Figure 6A-a). Meanwhile, C/EBPb expression was significantly lower in refractory and relapsed patients ( Figure 6A-b). Besides, the inverse correlation between c-Myc and C/EBPb mRNA level was observed in primary AML cells ( Figure 6A-c). Next, BMMCs from 5 AML patients were treated with 10058-F4, and flow cytometry was used to detect the differentiation state of primary AML cells. Our data showed that the percentage of CD11bpositive cells was increased after 10058-F4 treatment, illustrating that inhibition of c-Myc expression promoted the primary AML cells to differentiate to mature cells ( Figure 6B). In the meantime, the cytotoxic effect of 10058-F4 was detected by MTT assay, and    Figure 6C). In addition, C/EBPb was up-regulated in primary AML cells treated with 10058-F4 ( Figure 6D). Therefore, 10058-F4 could induce differentiation and retard the proliferation in primary AML cells.

Discussion
Here, we addressed that up-regulation of c-Myc expression was one of cytogenetic characteristics in the drug-resistant leukemic cells with increased colony formation capacity ( Figure 1). Overexpression of c-Myc in NB4 cells reduced the drug sensitivity to Ara-C, DNR or Doxo, and enhanced the capacity of colony formation ( Figure 2). Inversely, inhibition of c-Myc by shRNA or specific c-Myc inhibitor 10058-F4 rescued the sensitivity to Ara-C, DNR or Doxo treatment in drug resistant cells and reduced the colony formation (Figure 2, 5). In the ATRA-induced differentiation model, c-Myc over-expression reduced CD11b expression, and exhibited a decreased reduction of NBT to formazan (Figure 3), which was associated with C/EBPb down-regulation ( Figure 4). Importantly, in primary AML BMMCs, the expression of c-Myc and C/EBPb displayed a strong negative correlation. 10058-F4 showed anti-proliferative activity and increased cellular differentiation with up-regulation of C/EBPb expression (Figure 6). These results clearly indicated an important role of c-Myc in the drug resistance of AML and demonstrated that c-Myc inhibition could overcome drug resistance in AML treatment.
Several studies reported that high c-Myc expression promoted tumor formation and development in vitro and in vivo while its down-regulation caused tumor cell growth arrest, enhanced  apoptosis, senescence, or differentiation [28]. Importantly, c-Myc expression contributed to leukemogenesis and promoted the leukemia disease progress [13]. c-Myc inhibition prevented leukemia initiation and impaired the growth of relapsed pediatric T-ALL cells [29]. Clinical studies reported that AML patients had significantly higher c-Myc expression than health controls [16]. Our finding showed that the drug-resistant leukemic cells displayed high c-Myc expression with increased colony formation capacity, as shown in Figure 1, which suggested that high level of c-Myc expression might predict poor prognosis. c-Myc over-expression reduced the sensitivity of NB4 cells to Ara-C, DNR or Doxo, and increased cell colony formation ability, while c-Myc inhibition in NB4-R2 cells restored the sensitivity to Ara-C, DNR or Doxo treatment and reduced the colony formation capacity ( Figure 2). Thus, c-Myc expression was positively correlated with drug resistance of leukemic cells, and could act as a significant clinical biomarker for AML prognosis. As a key transcriptional factor, c-Myc decides the cell fate through regulation of a series of downstream genes. c-Myc regulates 15% of all genes expression through binding on Enhancer Box sequences (E-boxes) and recruiting histone acetyltransferases (HATs) [30,31]. Previous study showed that c-Myc determined transcriptional profiles of ATP-binding cassette (ABC) transporter genes in CML CD34+ hematopoietic progenitor cells, leading to drug efflux and resistance [32]. As well, c-Myc regulated multiple microRNAs, including induction of microRNAs with oncogenic properties and repression of microRNAs with tumor suppressor function. c-Myc increased miR-144/451 expression, contributing to the acquired imatinib resistance in K562 cells [33]. In addition, c-Myc antagonized the differentiation induced by imatinib in CML cells [11]. Of importance, low stage of cell differentiation was associated with poor outcome in tumors [34]. Using the ATRA-induced differentiation model, c-Myc overexpression in NB4 cells maintained poor differentiation, as presenting with decrease of CD11b expression and NBT reduction ( Figure 3). Moreover, c-Myc inhibitor 10058-F4 increased CD11b expression in the AML patient BMMCs (Figure 6). These results indicated that c-Myc expression was associated with drug resistance of AML cells through maintaining the state of poor differentiation.
With the characterization of the interplay between c-Myc and downstream targets, the mechanism of c-Myc maintains cell immature state still needs to clarify. Myc down-regulated C/EBP transcription factors was reported previously [35]. It has been shown that c-Myc repressed C/EBPa [36], and interacted with Max and Miz1 to suppress C/EBPd promoter activity and gene expression [37]. In our study, we found that c-Myc overexpression inhibited the expression of C/EBPb induced by ATRA treatment. C/EBPb over-expression could reverse c-Myc induced drug resistance (Figure 4). Importantly, the expression of c-Myc was negatively correlated with C/EBPb in primary AML cells ( Figure 6). Thus, AML cells might maintain immature or poorly differentiated state to prevent cytotoxicity through C/EBPb repression by c-Myc. Inhibition of c-Myc in AML could help to reverse the drug resistance of leukemic cells and enhance the treatment efficacy.
The main reason for the poor five-year survival rate in AML patients was that leukemic cells displayed drug resistance to the current drugs, leading to disease relapse. Thus, new approach in AML treatment with specific inhibitor targeting drug resistance related genes was required to reduce AML relapse. Previous reports showed that c-Myc inhibition sensitized the chemotherapeutic agents against different types of tumors, including liver cancer [21], Lewis lung carcinoma [18] and melanoma [38]. In our study, c-Myc inhibitor 10058-F4 promoted AML cells differentiation through increasing the rate of CD11b positive section and restored cell sensitivity to Ara-C, DNR or Doxo ( Figure 5, 6), indicating that inhibition of c-Myc activity could suppress the drug resistance to chemotherapeutics in AML cells by promoting cell differentiation. Currently, CX-3453 targeting c-Myc was now in phase II clinical trials (NCT00780663) for neuroendocrine carcinoma. With the wide use of c-Myc inhibitor for different cancers in clinical trials, it should be considerate to apply c-Myc inhibitor in combination therapies to overcome drug resistance, reducing disease relapse.
In conclusion, we found that c-Myc over-expression in AML cells reduced the sensitivity to chemotherapeutic drugs. Inhibition of c-Myc could rescue drug sensitivity by increasing C/EBPb expression. Taken together, c-Myc inhibition provided a novel strategy to overcome drug resistance in AML treatment. Figure S1 NB4-R2 and K562/G cells are respectively resistant to ATRA and imatinib. (A) NB4 and NB4-R2 cells were treated with various concentrations of ATRA for 72 h, and MTT assay was performed. (B) K562 and K562/G were exposed to various concentrations of imatinib for 48 h, and the cell viability was also tested by MTT assay. ***p,0.001, Student's t test.