Background
Chronic Myeloid Leukaemia (CML) is a clonal myeloproliferative disorder caused by the constitutive tyrosine kinase activity of the BCR/ABL1 fusion protein, the product of the Philadelphia (Ph) chromosome, generated from the t(9;22)(q34;q11) translocation [
1]. If untreated, CML progresses within 3-5 years from a mild and easy to control form, called chronic phase (CP), to the aggressive and incurable blast crisis (BC), the final phase of this disease. In CP, BCR/ABL1 expression induces a survival advantage but leukemic cells hold their capacity to differentiate normally. Conversely BC, as any acute leukemia, is marked by a complete differentiation block and by the ensuing accumulation of blasts.
At the molecular level BC is a heterogeneous disease [
2,
3]. Regardless of the additionally secondary changes, one common feature during the evolution from CP to BC is a marked increase in BCR/ABL1 expression [
3]. Progression to BC possibly occurs as a result of this increase, which leads to the activation of several events in primitive progenitor’s cell, such as genomic instability, acquisition of resistance to apoptosis, and activation of beta catenin in granulocyte-macrophage progenitors resulting in the acquisition of self-renewal capacity [
4‐
8]. Although the biological consequences of BCR/ABL1 up-regulation have been intensively studied, the molecular mechanisms responsible for this increase in expression are mostly unexplored.
After the oncogenic translocation, the BCR/ABL1 gene is under the transcriptional control of the BCR promoter, which may play a critical role in controlling BCR/ABL1 expression [
9]; in fact a similar dysregulation of both BCR and BCR/ABL1 gene transcription is evident in BC [
10]. Limited efforts have been devoted until now to the characterization of BCR promoter: according to Shah et al. [
9] a functional promoter is localized in a region of 1Kb at the 5′ of the BCR exon 1 coding sequence. Recent results from Marega et al confirmed the importance of this region for BCR basal transcriptional level: this study showed that the sequence comprised between -1443 and -1202 bp from the ATG site is critical to achieve a high level of
BCR promoter activity [
10].
MYC is a transcription factor belonging to the basic-helix-loop-helix-leucine zipper (bHLH-LZ) family [
11]. It forms heterodimers with the bHLH-LZ partner MAX, subsequently binding to a core DNA consensus region. Assembly of MYC/MAX heterodimer and DNA-binding seems to be crucial for the mitogenic, oncogenic and antiapoptotic functions of MYC [
12,
13]
. MYC is activated in various cancers through different genomic events including chromosomal translocation or gene amplification [
14‐
18]. MYC protein has been shown to play a role in BCR/ABL1 mediated transformation, mainly by acting as a cooperative oncogene with the fusion protein [
19‐
21]. Furthermore, BCR/ABL1 can induce MYC activity through distinct mechanisms: by regulating MYC expression through PI3K, JAK2 pathways and the E2F1 transcription factor [
22‐
24], by inhibiting MYC proteasome-dependent degradation through activated JAK2 [
25] and by regulating MYC mRNA translation by enhancing HNRPK translation-regulation activity [
26]. MYC is also a known BCR binding partner [
27] and higher BCR levels can decrease MYC protein levels, thus suggesting that BCR monoallelic disruption through BCR/ABL1 translocation may contribute to MYC protein stability in CML. Interestingly, MYC expression is normal in CP-CML, but is frequently up-regulated in BC through chromosome 8 amplification or over-expression [
28]. Recently, Lucas CM et al. [
29] showed that pharmacological inhibition of MYC reduces BCR/ABL1 tyrosine kinase activity and its expression level. These data suggest a causative role for MYC in the regulation of BCR/ABL1 expression, but they did not identify the molecular basis of this phenomenon. In the present study we demonstrate for the first time that MYC/MAX heterocomplex binds to the BCR promoter at four specific binding sites, leading to up-regulation of BCR and BCR/ABL1 at both transcriptional and protein levels in CML cell lines. Accordingly, silencing of MYC expression in various BCR/ABL1 positive cell lines causes significant downregulation of BCR and BCR/ABL1, decreases proliferation rate and induces cell death in CML cells. Since MYC is frequently over-expressed in BC, this phenomenon could be responsible for the BCR/ABL1 up-regulation and blast aggressiveness showed during CML evolution.
Discussion
In CML cells, after the t(9;22)(q34;q11) translocation and the formation of the Philadelphia chromosome, the BCR promoter drives the production of the BCR/ABL1 mRNA [
9]. This oncogenic product is necessary and sufficient for the malignant transformation of hematopoietic cells
in-vitro and
in-vivo [
33] and during CML progression the BCR/ABL1 oncogene persist and its level increases [
34‐
36]. The enhanced BCR/ABL1 activity characteristic of BC phase, seems to influence proliferation, survival, genetic instability and differentiation of myeloid progenitors [
3,
5‐
8].
A number of previous reports suggested the involvement of MYC in the clonal evolution of CML. MYC protein collaborate with BCR/ABL1 to induce blastic transformation, as assessed by the therapeutic efficacy of a silencing combination therapy for MYC and BCR/ABL1 of primary CML cells in SCID mice [
37]. Interestingly, MYC is also often upregulated in BC through chromosome 8 trisomy or gene amplification [
19,
20,
28,
38]. It has also been suggested that BCR can decrease MYC activity by regulating its stability at protein level [
27]: BCR disruption during BCR/ABL1 translocation can thus contribute to CML transformation leading to MYC upregulation. MYC levels are also controlled by BCR/ABL1 oncoprotein at transcriptional and protein levels [
22‐
26]. The role of MYC activity in blastic transformation is also strengthen by the fact that MYC is a known beta-catenin target gene, which has been shown to be activated in BC patients [
4,
39].
According to previous reports, BCR is physiologically down-regulated upon myeloid maturation from hematopoietic stem cells (HSCs) to common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and this mechanism is conserved in healthy donors and in CP-CML [
39]. Conversely, in BC this regulation is impaired for both BCR/ABL1 and BCR, which suggests the presence of ‘
in trans’ deregulated transcription of both BCR and BCR/ABL1 promoters associated with CML progression [
10]. A direct effect of MYC leading to an increase in BCR/ABL1 levels has recently been suggested [
29], but the molecular mechanism responsible for this phenomenon has not been clarified.
We showed here that MYC/MAX heterodimer can bind to BCR promoter at four specific loci (Fig.
1), thus regulating BCR and BCR/ABL1 expression in several CML cell line models (Figs.
2 and
3). By using a luciferase reporter assay we confirmed that MYC can modulate BCR and BCR/ABL1 expression by directly controlling BCR promoter activity (Fig.
5). Interestingly, when PBS3 and PBS4 were deleted from the BCR promoter construct (Full_BCR ∆3,4; 1200_BCR ∆3,4), a dramatic decrease in luciferase activity was observed when compared to all the other constructs, thus suggesting a critical role of these two regions in the regulation of BCR promoter activity (Fig.
5).
Our results show the existence of a positive feedback mechanism between the fusion protein BCR/ABL1 and the MYC transcription factor. BCR/ABL1 expression increases MYC activity which, in turn, is able to up-regulate BCR/ABL1 levels through direct binding on BCR promoter.
Methods
Cell lines
The BCR/ABL-positive CML cell lines K562, LAMA-84 and KCL-22 (DSMZ, Braunschweig, Germany) were cultured in RPMI-1640 medium supplemented with 10 % FBS, 2mM L-glutamine, 100 U/ml penicillin G, 80 μg/ml gentamicin and 20 mM HEPES in a 5 % CO2 incubator at 37°C. The 293-FT (R700-07, Life Technologies-Thermo Fisher Scientific, Waltham, MA USA) and the 293 cell lines (R750-07, Life Technologies-Thermo Fisher Scientific) were maintained following manufacturer instructions.
In silico analysis
To define the transcription factors involved in the regulation of BCR expression,
in silico analysis of BCR promoter (1443bp from the ATG site) was done using the open access Jaspar core database [
40], available on
http://jaspar.genereg.net/. The putative binding sites for transcription factors were generated from the distributions of bases at each position of all the transcription factors frequency matrices available in the JASPAR database. The putative binding sites with a relative profile threshold ≥ 80 % and a binding score >8 were selected for subsequent analyses.
Chromatin Immunoprecipitation (ChIP)
Chromatin immunoprecipitation was performed on K562 cells as previously reported [
41]. Briefly, proteins were cross-linked with 0.4 % formaldehyde and cells lysed. Chromatin was fragmented with a Bioruptor sonicator system (Diagenode, SA, USA) and subsequently immunoprecipitated with anti-MYC[N-262] (SC-764X, Santa Cruz Biotechnology Inc., Texas, USA), anti-MAX[C-17] (SC-197X, Santa Cruz Biotechnology) or non-specific IgG antibodies. After purification, immunoprecipitated DNA was amplified with RT-qPCR as described in [
42]. Results were visualized after separating PCR products by agarose gel with ethidium bromide staining. Primers used for the quantitative RT-PCR were: BCR-MycMax-Fw
5′GGGAAGTTCTGAGTCAGGTCG
3′ and BCR-MycMax-Rw
5′TGAGTAACCAATGCCAGCACCC
3′ for the amplification of a region including PBS1 and PBS2; 2BCR-MycMax-Fw
5′GAGGACTGCTGCGAGTTCTGCC
3′ and 2BCR-MycMax-Rw
5′-GACTCCCTGGTCCATAAAGACC
3′ for the amplification of a region including PBS3 and PBS4; NPM1_MycFw
5′CTCGTGAGCCAGGGATGCT
3′ and NPM1_MycRw
5′CCCTAGTGCTACCAGCCTCTTAAC
3′ for the amplification of MYC binding positive control; MARS-MaxFw
5′AAGTGCGACTTGCCCTAAAA
3′ and MARS-MaxRw
5′CCATGCAGCTGGGACTACA
3′ for the amplification of MAX binding positive control [
31,
32].
Real-time quantitative PCR (RT-qPCR)
Total RNA was extracted by Trizol reagent following standard procedures (Life Technologies). cDNA was synthesized from 1μg of total RNA, using Reverse Transcription Reagents (Applied Biosystems-Life Technologies). The total RNA obtained from transfectant cells were pretreated with DNAseI (Life Technologies) to avoid contamination from genomic DNA. RT-qPCR was performed using TaqMan® Brilliant II QPCR Master Mix (Agilent technologies, Santa Clara, CA, USA) for TaqMan assays or with Brilliant III Ultra-fast SYBR Green QPCR Master Mix (Agilent technologies) for SYBR Green assays on a Stratagene-MX3005P (Agilent technologies) under standard conditions. All the RT-qPCR experiments were performed in triplicate. The housekeeping gene β-glucronidase (GUSB) was used as an internal reference as assessed by [
43]. For MYC and MAX expression analysis we used TaqMan® Gene Expression Assays(Applied Biosystems-Life Technologies) consisting of a pair of unlabeled PCR primers and a TaqMan® probe. TaqMan RT-qPCR was performed according to the manufacturer’s specifications. The assay identification numbers were as follows: MYC-Hs00153408_m1, MAX-Hs01105524_g1. BCR, BCR/ABL and GUSb RT-qPCR primers/probes have been previously described [
10].
Western blot analysis
Western Blot was performed as previously described [
42] using the following antibodies
: rabbit anti-MAX [C-17] (sc-197X)(Santa Cruz Biotechnology), mouse anti c-MYC[9E10] (sc-40)(Santa Cruz Biotechnology), rabbit anti-c-ABL[K-12] (sc-131)(Santa Cruz Biotechnology), rabbit anti PARP-1[E102] (ab32138)(Abcam, Cambridge, UK) and anti-ACTIN (A2066)(Sigma-Aldrich, St Louis, MO, USA).
pGL3_BCR promoter constructs were obtained as in [
10]. Briefly, a region of 1443bp of the human BCR gene (NC_000022.11) from the first nucleotide upstream the ATG starting site was inserted in the pGL3 vector (Fig.
1b). Mutations in the BCR promoter at the MYC:MAX binding sites were introduced by the following protocol. Specific primers were designed and used to mutagenize the pGL3-Full_BCR and the pGL3-1200_BCR plasmids by using the Pfu Ultra High Fidelity enzyme (Agilent Technologies, Santa Clara, CA, USA). The products were then digested with DpnI (Roche, Indianapolis, IN, USA) and 2 μl were used to transform the competent TOP10 bacterial strain (Life Technologies). The presence of the deletion was confirmed by Sanger sequencing. The primer sequences used for mutagenesis reaction were as follows: MycMax-34-Fw
5′GAGGTAGGTGGTGGGGCTTGGCTGTTCCAGGACTGCAGGACTG
3′; MycMax-34-Rw
5′CAGTCCTGCAGTCCTGGAACAGCCAAGCCCCACCACCTACCTC
3′.
RNA interference
MYC silencing was generated infecting K562, LAMA-84 and KCL-22 cells with lentiviral particles obtained from modified FUGW lentiviral vectors: FUGW-MYC-shRNA(shMYC) kindly provided from Dr. RN Eisenman [
44]
, FUGW-H1-scrambled control shRNA (shNC) was a gift from Sally Temple [
45] (Addgene plasmid # 40625; Cambridge, MA, USA). Lentiviruses were packaged in 293-FT cells by co-transfecting the shRNA vectors with the packaging pCMV-dR8.91 and VSVG plasmids using jetPRIME Polyplus (Polyplus-transfection S.A, New-York, NY, USA) following manufacturer recommendations. Lentiviruses-infected cells were analyzed for GFP positivity with a a FACSAria flow cytometer (BD Bioscience, San Jose, CA, USA) and FACS-sorted when infection efficiency was lower than 85 %.
Generation of MYC and MAX expressing cell lines
MAX cDNA was obtained from total RNA of K562 cell line. Two primers spanning the whole MAX coding sequence (NM_145112.2) and introducing artificial HindIII and EcoRI sites at the 5′ and 3′ ends of the coding region were used for amplification and the PCR product was cloned into the pCDNA3 vector. Sequences of the primers were: MAX_Fw
5′AATAAAGCTTGAAATGAGCGATAACGATGAC
3′; Max_Rw
3′AATAGAATTCCCCGAGTGGCTTAGCTGGCCT
5′. pWZL_Blast_MYC vector was a gift from William Hahn [
46] (Addgene, plasmid # 10674). K562 cells were electroporated using a GenePulser XCell (Bio-Rad, Hercules, CA) (270 V, 975 μF). Transfected cells were selected with 5 μg/ml blasticidin and/or 1 mg/ml geneticin.
Luciferase assay
293 cells were infected with the lentiviral vectors encoding either MYC-shRNA(shMYC) or the scrambled-shRNA(shNC). RT-qPCR and Western Blot were used to confirm MYC down-regulation. Infected 293 cells were then co-transfected with pRL and pGL3/BCR promoter constructs prepared as described in Marega et al. [
10] and luciferase activity was determined after 72 h using the Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) and measured with the 1450 Wallac MicroBeta®luminometer (PerkinElmer, Waltham, Massachusetts, USA). All the experiments were performed in triplicates.
Cell viability assay
Cell viability was monitored by CellTiter 96 AQ One Solution Cell Proliferation Assay (Promega). The MTS tetrazolium reagent was added to the cells after 24h, 48h, 72h and 96h from seeding. Absorbance was assayed with a Wallac 1450 MicroBeta Trilux (PerkinElmer).
Cell cycle analysis
Cells were fixed in ethanol and stained with propidium-iodide (Sigma-Aldrich). Flow cytometry was performed on a Becton Dickinson FACSCalibur (Becton Dickinson Immunocytometry Systems, Mountain view, CA, USA) and data were analysed by FCS Express 4 Flow Research Edition software.
Statistical analysis
All the statistical analyses (unpaired two-tailed T-test) were performed by the GraphPad Prism (GraphPad, CA, USA) statistical package.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interest
The authors declare that they have no competing interest.
Authors’ contributions
NS performed the experiments and wrote the first draft of the paper; VM designed and performed the experiments, supervised work and wrote the final version of the paper; RGP designed the experiments and supervised work; CS MC, KP and PA performed experiments; CGP conceived and designed experiments,supervised work. All authors have read and approved the final manuscript.