C-MYC Oncoprotein Dictates Transcriptional Profiles of ATP-Binding Cassette Transporter Genes in Chronic Myelogenous Leukemia Cd34þ Hematopoietic Progenitor Cells

Published OnlineFirst June 21, 2011; DOI: 10.1158/1541-7786.MCR-10-0510

Molecular Cancer Cancer Genes and Genomics Research

c-MYC Oncoprotein Dictates Transcriptional Profiles of ATP-Binding Cassette Transporter Genes in Chronic Myelogenous Leukemia CD34þ Hematopoietic Progenitor Cells

Antonio Porro1, Nunzio Iraci1, Simona Soverini2, Daniel Diolaiti1, Samuele Gherardi1, Carolina Terragna2, Sandra Durante2, Emanuele Valli1, Thea Kalebic3, Roberto Bernardoni1, Chiara Perrod1, Michelle Haber4, Murray D. Norris4, Michele Baccarani2, Giovanni Martinelli2, and Giovanni Perini1

Abstract Resistance to chemotherapeutic agents remains one of the major impediments to a successful treatment of chronic myeloid leukemia (CML). Misregulation of the activity of a specific group of ATP-binding cassette transporters (ABC) is responsible for reducing the intracellular concentration of drugs in leukemic cells. Moreover, a consistent body of evidence also suggests that ABC transporters play a role in cancer progression beyond the efflux of cytotoxic drugs. Despite a large number of studies that investigated the function of the ABC transporters, little is known about the transcriptional regulation of the ABC genes. Here, we present data showing that the oncoprotein c-MYC is a direct transcriptional regulator of a large set of ABC transporters in CML. Furthermore, molecular analysis carried þ out in CD34 hematopoietic cell precursors of 21 CML patients reveals that the overexpression of ABC transporters þ driven by c-MYC is a peculiar characteristic of the CD34 population in CML and was not found either in the þ population of mononuclear cells from which they had been purified nor in CD34 cells isolated from healthy donors. Finally, we describe how the methylation state of CpG islands may regulate the access of c-MYC to ABCG2 gene promoter, a well-studied gene associated with multidrug resistance in CML, hence, affecting its expression. Taken together, our findings support a model in which c-MYC–driven transcriptional events, combined with epigenetic mechanisms, direct and regulate the expression of ABC genes with possible implications in tumor malignancy and drug efflux in CML. Mol Cancer Res; 9(8); 1054–66. 2011 AACR.

Introduction accounts for approximately 20% of all leukemias diagnosed þ in adults (1). In CML, the number of CD34 progenitor Chronic myeloid leukemia (CML) is probably the most cells represents a useful diagnostic and prognostic indicator regarding the evolution of this disease (2). Furthermore, in extensively studied human hematopoietic malignancy and þ CML, CD34 hematopoietic progenitors carry the t(9;22) Authors' Affiliations: 1Department of Biology; 2Institute of Hematology "L. (q34;q11) reciprocal chromosomal translocation (chromo- and A. Seragnoli", University of Bologna, Bologna, Italy; 3Novartis Oncol- some Ph), which gives rise to the BCR-ABL proto-oncogene ogy, Clinical Development, New Jersey; and 4Children's Cancer Institute Australia For Medical Research, New South Wales, Sydney, Australia and its constitutively active protein tyrosine kinase product Current address for A. Porro: Ecole Polytechnique Federale de p210/BCR-ABL (3). Currently, treatment of CML is based Lausanne, ISREC-Swiss Institute for Experimental Cancer Research, on the administration of the tyrosine kinase inhibitors, Lausanne, Switzerland. Current address for N. Iraci: Brain Repair which selectively inhibit BCR-ABL tyrosine kinase activity Centre & Cambridge Stem Cell Initiative, University of Cambridge, Cambridge, United Kingdom. Current address for D. Diolaiti: Fred responsible for the pathogenesis of this disease (4). How- Hutchinson Cancer Research Center, Division of Basic Sciences, Seat- ever, patients may experience resistance to tyrosine kinase tle, WA. Current address for T. Kalebic: Millenium, Takeda Company, inhibitors, thus limiting the long-term benefits of the drug. Cambridge, MA. Current address for C. Perrod: Max Delbruck€ Center for Molecular Medicine, Berlin, Germany. The most extensively studied mechanism of drug resistance Note: Supplementary data for this article are available at Molecular Cancer is represented by point mutations in the BCR-ABL kinase Research Online (http://mcr.aacrjournals.org/). domain that impair tyrosine kinase inhibitors binding A. Porro and N. Iraci contributed equally to the work. (reviewed in ref. 5). Nonetheless, mutations have been Corresponding Author: Giovanni Perini, Department of Biology, Univer- found to mediate resistance only in a proportion of resistant sity of Bologna, via F. Selmi 3, 40126 Bologna, Italy. Phone: 39-051-209- patients. Other mechanisms have been invoked but very 467; Fax: 39-051-209-4286; E-mail: [email protected] or Giovanni little is known of their actual role. In this regard, the Martinelli, Molecular Biology Unit, Department of Hematology/Oncology "Seragnoli", University of Bologna, Via Massarenti, 9, 40138 Bologna, Italy. intracellular concentration of tyrosine kinase inhibitors is Phone: 39-051-636-3829; Fax: 39-051-636-4037; E-mail: a critical feature influencing drug efficacy. Successful ther- [email protected] apy of CML is impeded by the development of resistance to doi: 10.1158/1541-7786.MCR-10-0510 a wide spectrum of chemotherapeutic drugs. In a number 2011 American Association for Cancer Research. of cancers, a drug-resistant phenotype has been linked to

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c-MYC Dictates ABC Expression in CML

overexpression of some members of the highly conserved characterized bythepresence ofdoubleminutechromosomes family of transmembrane proteins characterized by an ATP- that contain MYC amplification (19, 20), and a recent study binding cassette (ABC) domain, the so called ABC super- suggests that several oncogenes, involved in myeloid tumor family of transporters (6, 7). Some studies have shown that progression, induce leukemogenesis by activating c-MYC the ABCB1 (PgP/MDR1) and ABCG2 members of this oncoprotein (21). Moreover, in CML, some studies have family are deregulated in imatinib-resistant cell lines and/or shown that BCR-ABL can indirectly activate c-MYC func- patients (8, 9). Less is known on the role of the ABC tion via either the Janus-activated kinase (JAK2) pathway transporters in mediating resistance to the second-generation (22) or the mitogen-activated protein kinase/heterogeneous tyrosine kinase inhibitors (dasatinib and nilotinib) approved nuclear ribonucleoprotein K (MAPK/HNRPK) pathway for imatinib-resistant or intolerant CML patients (10–13). (23) causing increased c-MYC mRNA translation. Further- Moreover, besides their role in conferring drug resistance, a more, c-MYC is located on chromosome 8 (8q24), and wide range of observations and correlative studies indicate trisomy of chromosome 8 is one of the most frequent that high levels of ABC transporters in tumors may determine additional abnormalities detected in CML patients (24). a malignant progression and a more aggressive phenotype Overall,thesefindingspointtoc-MYCasamaindownstream independentlyoftheirroleindrugefflux(reviewed inref.14). actor in leukemias, and in particular in CML pathogenesis, Misregulation of the ABC transporter genes in CML may possibly by modulating transcription of a large set of genes. occur through activity of specific transcription factors whose Recently, we have established that MYCN is responsible function is also altered in this disease. MYC dysregulation is for directing and coordinating the transcription of ABC considered an oncogenic event required for human tumor- genes in neuroblastoma tumors (25). Here, we have inves- igenesis (15–17) and has also been related to the progression tigated whether c-MYC can also orchestrate ABC transcrip- of myeloid leukemias (18). The proto-oncogene c-MYC tion in myeloid leukemias. Importantly, we found that c- þ encodes a basic helix-loop-helix leucine zipper transcription MYC is highly expressed in CD34 progenitor cells from factor that, dimerizing with its partner MAX, controls multi- newly diagnosed chronic phase (CP)-CML patients and that ple cell functions. Patients with myeloid leukemias are often its transcriptional level correlates with that of specific ABC

HL-60 Figure 1. c-MYC regulates ABHL-60 transcriptional activity of a large c-MYC group of ABC drug transporter c-MYC genes in HL-60 cells. A, top, β-Actin β-Actin modulation of c-MYC expression DMSO 0 h 24 h 48 h by DMSO in HL-60 cells was shRNA ctrl c-MYC c-MYC monitored at the protein level 10 d 12 d using Western analysis. Bottom, cluster 3 (28) was used to generate

an image map showing HL-60 0 h HL-60 24 h HL-60 48 h

ABC ABCA2 10 d 12 d expression levels of genes as ABCF2 a function of c-MYC ABCB10 HL-60 shc-MYC HL-60 shc-MYC downregulation mediated by ABCA4 HL-60 shctrl ABCE1 ABCB10 DMSO treatment. Green indicates ABCC1 ABCB9 genes positively regulated by c- ABCB9 ABCC4 ABCC1 MYC, whereas red indicates those ABCF3 negatively regulated. Genes, the ABCB4 ABCA2 expression of which is totally ABCB7 ABCF1 ABCF1 absent in HL-60 cell line, are listed ABCA10 ABCC4 below the cluster. B, top, ABCB8 ABCE1 modulation of c-MYC expression ABCC2 ABCD4 ABCF2 following infection of HL-60 with ABCC6 ABCA10 viruses expressing shRNA ctrl and ABCC10 ABCB2 shRNA c-MYC after 10 and ABCB6 ABCD2 ABCA7 12 days of selection with ABCD1 puromycin. Bottom, cluster 3 (28) ABCA9 ABCG1 ABCB2 was used to generate an image ABCA1 ABCD1 map showing expression levels of ABCC5 ABCD3 some ABC genes, selected from ABCD3 ABCA5 the previous analysis, as a ABCA7 –1.09 –0.00 +0.97 function of c-MYC ABCG1 downregulation mediated by –2.07 –0.00 +2.14 specific shRNA. Green indicates Not expressed:ABCA3, ABCA6, ABCA8, ABCA12, ABCA13, genes positively regulated by c- ABCB1, ABCB3, ABCB5, ABCB11, ABCC3, MYC, whereas red indicates those ABCC7, ABCC8 ABCC9, ABCC11, ABCC12, negatively regulated. ABCG2, ABCG4, ABCG5, ABCG8

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Figure 2. c-MYC binds multiple ABC transporter gene promoters in vivo. Quantitative crosslinking ChIP (qChIP) was carried out in HL-60 cells before (high c-MYC) treatment with DMSO. The relative fold enrichment was determined by comparing the enrichment of sequences immunoprecipitated with anti–c-MYC or anti-MAX antibodies with that obtained with a preimmune serum (IgG). Results represent the mean SE of 5 independent ChIP experiments in which each region was amplified by qRT-PCR in triplicate SE. Promoter diagram: bent arrow, transcription start site; grey arrow, canonical E-box; black arrow, noncanonical E-box; open boxes, amplicons indicated with a capital letter; chromosome and coordinates (bp) are also given.

transporter genes. Our results show that c-MYC directly with important implications for the treatment of different controls the transcription of a large subset of ABC types of leukemia. transporter genes in myeloid leukemia cells. Finally, we have investigated how, besides c-MYC–mediated transcrip- Materials and Methods tional events, epigenetic mechanisms can lead to dysregulation of ABCG2, a well-studied protein associated with Cell lines and patient samples multidrug resistance in CML. KG-1a cells were cultured in RPMI containing 20% Overall, c-MYC–driven transcription of ABC genes may FBS heat inactivated and 50 mg/mL gentamycin. HL-60 provide insights into the molecular mechanisms of cancer and K562 cells were cultured in RPMI medium 1640 drug resistance in CML and it may represent an unexpected containing 10% FBS heat inactivated and 50 mg/mL target for the development of novel therapeutic strategies gentamycin.

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Figure 2. (Continued).

Peripheral blood samples from 21 newly diagnosed and ligated into pCL (high titer) expression vector. For c- CP CML patients were collected after written informed MYC downregulation, we used the following targeting consent. Mononuclear cells were obtained by Ficoll–Hypa- sequence: 50-GATGAGGAAGAAATCGATG-30 pre- þ que density gradient centrifugation. CD34 cells were viously established in the laboratory of Professor Martin selected by using the human CD34 microbead kit on an Eilers (26). Retroviruses expressing c-MYC shRNA and automatic immunomagnetic separator (AutoMacs, Milte- nontargeting shRNA were produced in HEK293T cells after nyi Biotec) according to manufacturer's recommendations. cotransfection of pCL expression vector and pC-f ampho packaging vector. After 24 hours, supernatants containing Retroviral production and infection of plasmids the retroviruses are collected and filtered through a 0.45-mm expressing short hairpin RNAs pore size filter. Each supernatant was diluted in 2.5 volume To generate short hairpin RNA (shRNA) vectors for of complete medium. Polybrene (8 mg) per milliliter of c-MYC, hairpin-encoding oligonucleotides were annealed medium was added. One day before the infection, the

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HL-60 cells were seeded such to be at 50% of confluency on study were the following: immunoglobulin G (IgG; Santa the day of infection. On the day of the infection, the medium Cruz sc-2027), c-MYC (Santa Cruz N-262), and Max was replaced with medium containing retrovirus and poly- (Santa Cruz C-17). Primers for quantitative ChIP are listed brene. Thirty-six hours after infection, the HL-60 cells were in Supplementary Table SII. grown in the presence of 8 mg/mL puromycin to select for successful transfectants of the shRNAs. After 10 and 12 days Treatment of cells with 5-aza-20-deoxycytidine of selection, cells were harvested and analyzed for proteins Cells were seeded at a density of 0.5 106 cells/mL and and RNA expression levels. treated with 5 mmol/L of 5-aza-20-deoxycytidine (AZA) for 72 hours. Luciferase assay The pGL3-basic and Renilla-TK vectors were obtained Methyl-CpG Immunoprecipitation from Promega. Promoters of selected ABC transporter genes The methyl-CpG immunoprecipitation (mCIP) was car- were obtained using PCR and cloned into the pGL3-basic ried out as previously described (28). The DNA was vector as described previously (25). The vectors were obtained by using the Blood & Cell DNA culture midi transfected in HL-60 previously infected with retroviruses Kit (Qiagen) according to the manufacturer's instructions. expressing c-MYC shRNA and nontargeting shRNA. The Immunoprecipitation was carried out by incubating 15 mg transfection was carried out after 10 days of selection with of sonicated DNA with 5 mg of 5-methylcytosine mono- puromycin by using Lipofectamine LTX Reagent according clonal antibody (Calbiochem NA81). to manufacturer's recommendations. The activity of firefly or Renilla luciferase was measured after 48 hours with Southern blotting the Dual Luciferase Assay Kit (Promega) according to Genomic DNA (10 mg/lane) was completely digested instructions. with EcoRI, EcoRI/MspI, or with EcoRI/HpaII restriction enzymes (New England Biolabs) and loaded onto a 1% Gene expression analysis agarose gel. DNA was transferred on a positively charged RNA samples were prepared by using Tri-Reagent Hybond-XL membrane (Amersham) following a standard (Sigma) and treated with DNase (DNA-free, Ambion). alkaline transfer protocol (Current Protocols in Molecular Reverse transcription and PCR were carried out by using Biology, Wiley). DNA probe was amplified by PCR, gel Thermoscript reverse transcriptase PCR (RT-PCR) kit purified and labeled with a-32P-dCTP by using the Rad- (Invitrogen). Expression data were obtained both from Prime DNA Labeling System (Invitrogen) according to the real-time quantitative PCR (qRT-PCR). qRT-PCR was manufacturer's instructions. Hybridization and washes were carried out by using iQ SYBR Green Supermix and the carried out at 65C using standard procedures. iQCycler thermocycler (Bio-Rad). Primers used for qRT- PCR are described in Supplementary Table SI. Results þ Gene expression profile (GEP) was carried out on CD34 cell fractions obtained from peripheral blood of CML c-MYC is critical for expression of several ABC patients at diagnosis by using the Affymetrix HG-U133 transporter genes in promyeloid leukemia cells Plus 2.0 microarray platform following the manufacturer's To investigate the potential role played by c-MYC in recommendation. Raw data were normalized using the transcriptional regulation of ABC transporter genes in Robust Multichip Average (RMA) algorithm and filtered, myeloid leukemias, we used the promyelocytic leukemia and the expression data of ABC transporters and c-MYC were cell line HL-60. Although highly expressing Bcl-2, HL-60 extrapolated from the whole transcriptome. The same cells were not selected for enhanced resistance to cytotoxic þ approach was applied to the GEP data of normal CD34 drugs (29). HL-60 cells lack expression of the well-defined cells, obtained from peripheral blood of healthy donors human stem cell marker CD34 and they are characterized [Gene Expression Omnibus (GEO) database, accession by c-MYC proto-oncogene amplification. Moreover, c- number GSE12662]. The comparison between the 2 sets MYC overexpression can be turned off by treating cells DDC of data was carried out by using the t method, with the with dimethyl sulfoxide (DMSO; ref. 30). By using this cell GUSB (housekeeping gene) as internal reference and the model system, we examined the expression level of all 48 medians of each gene expression values as calibrators. human ABC drug transporters as a function of c-MYC silencing at 24- and 48 hours of DMSO treatment. Tran- Methylation-sensitive PCR scription profiles were determined by qRT-PCR and This protocol was carried out as described previously clustered by using the Cluster 3 program (31). Results (35). Genomic DNA was digested with EcoRI or PmlI and show that DMSO-mediated differentiation can affect then used for PCR. Primers are described in Supplementary transcription of a large subset of ABC transporter genes Table S2. (Fig.1A).Theanalysisreveals4groupsofABC transporters depending on the correlation of their expression levels Chromatin immunoprecipitation assays with that of c-MYC. For example, a high–c-MYC level Chromatin immunoprecipitation (ChIP) was carried out correlates with increased expression of a specific group of as previously described (27). Antibodies employed in this ABC genes including ABCA2, ABCB9, ABCB10, ABCC1,

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ABCC4, ABCE1, ABCF1,andABCF2,amajorityof which has been implicated in drug resistance. On the other hand, the expression of another group of ABC transporters such as ABCA7, ABCB2, ABCD1, ABCD3, and ABCG1 genes inversely correlated with expression of c- MYC. Two additional groups were also identified: one in which genes did not change their expression as a function of differentiation; and a second one in which genes were not at all expressed in HL-60 cells. To further determine whether c-MYC is required to activate or repress transcription of ABC genes and to rule out pleiotropic effects because of the use of DMSO, we have manipulated c-MYC expression directly and specifically by using a RNA interference approach. HL- 60 cells were infected successfully with nontargeting shRNA (shctrl) or c-MYC targeting shRNA (shMYC) retroviruses. The cells were selected in presence of puromycin, and the expression of the subset of ABC genes previously showed to be responsive to treatment with DMSO was monitored after 10 days and 12 days postselection. Results confirm that c- MYC plays a key role in regulating transcription of a large subset of ABC transporter genes (Fig. 1B). c-MYC is a direct transactivator of a large group of ABC transporter genes Next, we investigated whether c-MYC directly contacts promoters of specific ABC transporter genes thus affecting their transcription. We mainly focused on those genes that were positively regulated by c-MYC and likely responsible for increased drug efflux. To address this issue, we carried Canonical E-boxes out bioinformatic analysis of the ABC promoter regions and shRNA ctrl shRNA c-MYC Noncanonical E-boxes found out that many of the MYC responsive ABC gene 12 d promoters contain several canonical and noncanonical MYC binding sites (E-box sites), the majority of which Figure 3. The role of c-MYC in ABC gene transcription is recapitulated are located in regions from 2,000 to þ2,000 base pairs by transient transfection assays. ABC transporter promoters were around the predicted transcription start site (Fig. 2). To cloned into a luciferase reporter vector (luc-reporter). Constructs were confirm that c-MYC indeed binds these promoters in vivo, tested in HL-60 cells as a function of c-MYC expression (shRNA ctrl and shRNA c-MYC). The ABCA10 gene was used as a negative control. we carried out ChIP on HL-60 cells. Results show that A renilla reporter cotransfected with each ABC-Luc reporter was used to promoters of ABCA2, ABCB9, ABCB10, ABCC1, ABCC4, normalize luciferase activity. Promoter diagram: bent arrow, transcription ABCE1, ABCF1, and ABCF2, were occupied by the c- start site; cloned DNA region (bp) is indicated below promoter map. MYC/MAX complex (Fig. 2). As a control, ChIP analysis Results are the mean SE of 3 independent transfections. was also employed on HL-60 cells pretreated with DMSO for 3 days, thereby, depleted of the c-MYC protein and results show that for each of these gene promoters (ABCA2, found that MAX but not c-MYC was associated with gene ABCB9, ABCB10, ABCC1, ABCC4, ABCE1, ABCF1, and promoters (Supplementary Fig. S1). As additional controls, ABCF2), luciferase activity was indeed dependent on c-MYC ChIP was carried out for ABCA10 that does respond to c- expression. The promoter of ABCA10 transporter gene MYC, for ABCB1 and ABCG2 that are not expressed in was cloned and used as negative control (Fig. 3). Overall, HL-60 cells and for APEX-1, a well-known MYC positive these data support the view that c-MYC may function as a target gene (Fig. 1). direct transactivator of a large subset of ABC transporter Because direct binding of c-MYC to gene promoters is genes. not per se sufficient to prove its effect on the transcription of target genes (32), we carried out experiments by using c-MYC controls expression of ABC transporters in þ luciferase reporter constructs carrying the promoter regions CD34 s hematopoietic progenitors of CML patients of those ABC transporters bound by c-MYC in ChIP The expression profiles of c-MYC responsive ABC trans- þ experiments. Recombinant reporters were transiently porter genes was analyzed by qRT-PCR in CD34 pro- cotransfected into HL-60 cells, and luciferase activity was genitors cells versus mononuclear cells of 21 newly quantified as a function of c-MYC downregulation, obtained diagnosed CP CML patients. Results show that the ABC through an shRNA strategy and DMSO treatment, respec- genes, whose promoters are bound by c-MYC, were highly þ tively (Fig. 3 and Supplementary Fig. S2). As expected, expressed in the CD34 cell population when compared

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Figure 4. c-MYC controls expression of ABC drug transporters in CD34þ hematopoietic progenitors. A, relative expression of c-MYC and ABC genes in the CD34þ cell population was compared with that of the entire population of mononuclear cells (MNC). Each sample was analyzed in

duplicate and threshold cycle (Ct) values were averaged. Results are reported as delta Ct values which were calculated as the difference between Ct þ of the ABC mRNAs and Ct of the GUSB mRNA used for normalization. B, c-MYC and ABC gene expression of CML CD34 cells was compared with þ that of CD34 cells from healthy donors. The 2 sets of data were compared by using the DDCt method, with the GUSB (housekeeping gene) as internal reference and the medians of each gene expression values as calibrators. Both in (A) and (B) significant differences between the 2 cell populations were determined by using Mann–Whitney statistical tests.

with the population of mononuclear cells from which they significantly higher levels of c-MYC and ABC transporter þ have been purified, and that their expression strongly genes by comparison with healthy donor CD34 cells correlated with that of c-MYC (Fig. 4A). Furthermore, (Fig. 4B). With regard to ABCG2, we recapitulated the the GEPs of c-MYC and the ABC transporter genes were same finding observed for those ABC genes selected as extrapolated from the whole transcriptome and compared positively regulated by c-MYC. Indeed, we observed higher þ þ with those of a CD34 cell population obtained from ABCG2 expression levels in the CML-CD34 cells than in healthy donors peripheral blood lymphocytes (data obtained the mononuclear population (Fig. 5A). Interestingly, from GEO, accession number GSE12662). The median because ABCG2 promoter is regulated by epigenetic expression values of c-MYC and each ABC transporter gene mechanisms, such as DNA methylation (33, 34), by using þ shows that the CML CD34 cell population expresses methylation-sensitive PCR, we found that the ABCG2

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Figure 5. c-MYC controls expression of ABCG2 in CD34þ hematopoietic progenitors. A, relative expression of ABCG2 genes in the CD34þ cell population was compared with that of the entire population of mononuclear cells (MNC). Each sample was analyzed in duplicate, and threshold cycle (Ct) values were averaged. Results are reported as delta Ct values which were calculated as the difference between Ct of the ABC mRNAs and Ct of the GUSB mRNA used for normalization. B, ABCG2 E-Box methylation status of CD34þ cells purified from a representative group of 10 patients affected by CML. C, ABCG2 gene expression of CML CD34þ cells was compared with that of CD34þ cells from healthy donors extrapolated from GEO database (left) or collected from 10 new healthy donors (right). Significant differences between cell populations were determined by using Mann–Whitney statistical tests. promoter was methylated on the MYC binding site, located share several biological and biochemical features with mye- þ – þ in close proximity to its transcriptional start site, in CD34 loid leukemia cells. K562 derives from a CD34 Ph CML, þ cells of 5 patients out of the 21 ones that were analyzed whereas KG-1a derives from a CD34 AML. Results show (Fig. 5B). Based on this observation, we examined whether that, in these cells, c-MYC can bind the promoter of all of there may be a correspondence between the methylation data the ABC transporter genes found to be positively regulated and the expression level of ABCG2 in our patient samples, by c-MYC, fostering the view that c-MYC plays a crucial and we found that indeed in those patients showing a role in ABC transporter expression in myeloid leukemias methylated MYC binding site, the expression of ABCG2 (Fig. 6). In contrast, no binding was observed for ABCB1 is the lowest (data not shown). Considering the above, we or ABCA10, neither of which had been shown to be have re-analyzed microarray data expression of ABCG2 only regulated by c-MYC. in those patients that carried an unmethylated ABCG2 geneandcomparedthosedatawiththoseofhealthy c-MYC–mediated transcription of ABCG2 gene is donors (data from GEO database). By doing so, the P controlled by its CpG island–promoter methylation value was definitely improved although it did not reach status statistical significance (Fig. 5C, left). Indeed, this trend ABCG2 is usually aberrantly overexpressed on primitive þ was then confirmed as statistically significant when CML CD34 HSCs. Indeed, it should be noted that in ABCG2 expression was measured by qRT-PCR, a more contrast to what has been found in HL-60 and K562 cells, þ sensitive technique than microchip arrays. In that case, ABCG2 was expressed both in CD34 progenitor cells as þ expression of ABCG2 in CML-CD34 cells was com- well as in KG-1a cells, and its expression correlated with þ þ pared with that of CD34 cells obtained from the per- that of c-MYC in CD34 progenitors of CML patients ipheral blood of 10 new healthy donors (Fig. 5C, right). (Fig.5A).Nonetheless,c-MYCwasfoundassociatedwith þ Next, we determined the binding of c-MYC to the ABC the ABCG2 promoter in KG-1a CD34 cells but not in þ transporter gene promoters in CD34 cells by carrying out HL-60 and K562 cell lines (Fig. 6), thus revealing a ChIP assay. Ideally, ChIP should have been carried out on complex pattern of mechanisms involved in controlling þ CD34 hematopoietic stem cells (HSC) of patients; how- ABCG2 transcription. One possible explanation of these ever, this was not possible because of the limited amount of findings may rely on the evidence that the promoter of þ CD34 HSCs that can be purified from peripheral blood or ABCG2 is often hypermethylated in cancer (33, 34). bone marrow of donor patients. To overcome this problem, Moreover, we have previously shown that MYC cannot ChIP was carried out on K562 and KG-1a cell lines, which bind its cognate sites when methylated (35). Therefore, in

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Figure 6. c-MYC is physically associated with promoters of ABC transporter genes in K562 and KG-1a cell lines. Quantitative crosslinking ChIP (qChIP) was carried out in K562 and KG-1a cell lines. The relative fold enrichment was determined by comparing the enrichment of sequences immunoprecipitated with anti–c-MYC or anti-MAX antibodies with that obtained with a preimmune serum (IgG). Results represent the mean SE of 5 independent qChIP experiments in which each region was amplified by qRT-PCR in triplicate.

the context of promoter methylation, c-MYC does not associate with the ABCG2 promoter after cells were treated have access to the ABCG2 promoter and thus cannot drive with AZA. Unfortunately, we found that AZA treatment ABCG2 transcription. In support of this hypothesis, it is downregulates MYC expression (data not shown), thus worth noting that in the HL-60 cell line, c-MYC affects technically preventing the execution of the ChIP assay. the luciferase activity of a reporter construct carrying the However,westillfeltthatdeterminingtheglobal unmethylated promoter of ABCG2,butcannotmodulate methylation status of the ABCG2 promoter would be its endogenous expression (Fig. 7A). To better investigate helpful to show that an epigenetic event such as hyper- whether a distinct promoter DNA methylation pattern methylation of ABCG2 promoter region is crucial for its may explain the different ABCG2 transcriptional levels, silencinginsomeleukemiccelllines.Todothat,the KG-1a, HL-60, and K562 cells were treated with AZA, a methylation status of the ABCG2 gene in KG-1a, HL-60, DNA demethylating agent, and mRNA expression of andK562cellswasdeterminedbymCIP,byusinga ABCG2 was monitored by qRT-PCR. Despite the fact monoclonal antibody that specifically recognizes the that 3 cell lines show same sensitivity to the treatment methylated cytosines. As shown in Fig. 7C, the putative with AZA (Supplementary Fig. S3) only in HL-60 and CpG island surrounding the transcription start site of K562 cells AZA can reverse transcription silencing of the ABCG2 promoter was found to be enriched in methylated endogenous ABCG2 gene, suggesting that lack of MYC cytosines in HL-60 and K562 cell lines compared with a responsiveness was most likely caused by promoter hyper- distal region. As expected, the same region was found to be methylation (Fig. 7B). Based on this assumption, we have completely unmethylated in KG-1a cells (Fig. 7C). To attempted to determine by ChIP, whether MYC can confirm these results, the DNA methylation state of the

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Figure 6. (Continued).

ABCG2 promoter was evaluated in HL-60 and K562 cells Discussion by Southern blot (Supplementary Fig. S4). Genomic DNA extracted from these 2 cell lines was digested with Resistance to chemotherapeutic agents is a major obstacle either the MspIorHpaII restriction enzyme and then for the successful treatment of cancer. The failure of the hybridized with a DNA probe corresponding to the curative treatment of cancer patients often occurs as a result ABCG2 promoter. Although HpaII and MspIrecognize of intrinsic or acquired drug resistance of tumors to che- the same CCGG sequence, HpaII can cleave that sequence motherapeutic agents. Therefore, understanding how che- only when unmethylated. As shown in Supplementary moresistance develops and eventually how it can be Figure S4, the hybridization profile reveals that the prevented, becomes crucial to fighting cancer effectively. ABCG2 promoter region is resistant to cleavage with Functional redundancy in the ABC transporters family HpaII but not to the cleavage with MspI, indicating that complicates their utility as therapeutical targets. In this the ABCG2 promoter region is highly methylated in HL- study, we have provided lines of evidence that c-MYC 60 and K562 cell lines. can target a wide spectrum of ABC transporter genes with Overall, the combined ChIP data along with the methy- different structural and functional features raising questions lation data allow us to conclude that c-MYC can target as to how their altered expression may be relevant for drug ABCG2 expression only when its cognate sites located efflux and more in general for tumor biology. In particular, within ABCG2 promoter are unmethylated and impor- our data suggest that c-MYC may contribute to the multi- tantly, in the context of different types of leukemia, this drug resistance profile and malignant progression of myeloid event seems to occur prevalently, if not exclusively, only in tumors by dysregulating the transcription of specific ABC þ þ CD34 progenitor hematopoietic cells as shown in Fig. 5B. transporter genes prevalently in CD34 hematopoietic

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Figure 7. Methylation status of the ABCG2 promoter. A, ABCG2 transporter promoters were cloned into a luciferase reporter vector and its activity was tested in HL-60 cells as function of c-MYC downregulation. B transcriptional reactivation of ABCG2 following the treatment of HL-60 and K562 cells with 5 mmol/L AZA for 72 hours. ABCG2 mRNA expression was determined by qRT-PCR and compared between control (–AZA) and treatment (þAZA). Results are expressed as fold difference between control and þAZA condition and setting to 1 the expression of ABCG2 in the control condition. C, mCIP was employed in KG-1a, HL-60, and K562 cell lines to determine the methylation status of the ABCG2 promoter in vivo. Position of the putative CpG island is shown schematically with the ABCG2 gene diagram (left). A and B represent a region distal from the transcription start site and the putative CpG island surrounding the transcription start site (TSS), respectively. Relative enrichment obtained with the specific antibody was compared with that obtained with preserum (IgG) which was set to 1 in the graph. Results are the average of 4 independent dual ChIP experiments.

progenitors of CML patients. The fact that BCR-ABL has tissue invasion, and metastasis (reviewed in ref. 14). Indeed, been previously shown to increase c-MYC expression (22, ABCC4 is involved in human dendritic cell migration (39) 23) and that c-MYC in turn coordinates transcription of a and its expression is affected during hematopoietic stem cell large set of ABC transporter genes, reveals a novel role of differentiation (40), whereas ABCF2 has been found ampli- BCR-ABL in potentially mediating drug efflux in CML. fied in ovary and breast cancers suggesting a possible role in Numerous studies have shown that members of the ABC malignant progression (41). Likewise, ABCG2 is highly family shown here to be positively regulated by c-MYC may expressed in a wide range of stem cells (reviewed in ref. þ be involved in the development of a chemoresistance phe- 42) and in primitive hematopoietic CML CD34 progeni- notype. ABCC1, ABCC4, and ABCG2 can confer resistance tors (9). ABCG2 is responsible to establish the side popula- to several antineoplastic drugs such as antifolates, doxoru- tion phenotype of HSCs (43), blocking hematopoietic bicin, and nucleotide analogs (36). In addition, the ABCA2 development, and maintaining the stem cell self-renewal transporter has been associated with drug resistance in potential. It has been shown that BCR-ABL TK inhibitors childhood T-cell acute lymphoblastic leukemia (37), exhibit high affinity interaction with ABCG2 in HSCs (44, whereas the ABCE1 gene, an inhibitor of Ribonuclease L, 9, 12). Although a considerable debate is ongoing about the was amplified in several tumorigenic cell lines and its high role played by ABCG2 in mediating imatinib resistance, a expression correlated with resistance to macrolide inhibitors recent study shows that ABCG2-transduced K562 cells are of protein synthesis (38). Furthermore, ABC transporters protected from imatinib-induced cell death, suggesting that play specific roles in cancer biology beyond the efflux of ABCG2 overexpression can indeed trigger resistance to cytotoxic drugs, contributing to some of cancer hallmarks, tyrosine kinase inhibitors in CML stem cells (12). In support such as evasion of apoptosis, limitless replicative potential, of this evidence, it has also been shown that overpression of

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c-MYC Dictates ABC Expression in CML

ABCG2 in HEK293 cells results in a reduced intracellular which express a variety of drug resistance mechanisms, imatinib accumulation (45) and in addition, in vivo studies survive and can repopulate the tumor (48). Therefore, have confirmed that imatinib is a substrate for transport by understanding the molecular bases for drug resistance of ABCG2 (46, 47). It has been previously reported that in HSCs may help devise novel therapeutic interventions human renal carcinoma as well as in multiple myeloma cell aimed at a complete eradication of minimal residual lines, alterations in the methylation state of the chromatin- disease. Secondly, upregulation of the ABC genes in the embedded ABCG2 promoter have a dramatic effect on its HSC population may predispose cancer cells to develop- expression profile (33, 34). Here, we provide a rationale of ing drug resistance following chemotherapy treatment, how epigenetic control of c-MYC–mediated transcription particularly when a low dosage of the drug is employed, þ can lead to ABCG2 upregulation in CD34 CML stem cells which may facilitate positive selection and expansion of but not in committed progenitor and mature blood cells. cells with high drug efflux capacity. Thus, a combination of transcriptional and epigenetic In conclusion, our study unveils novel aspects of the BCR- mechanisms may explain the altered expression of ABCG2, ABL/c-MYC cross-talk that may have important implica- and more in general of ABC transporter genes, hence con- tions for drug resistance and risk assessment in CML. ferring peculiar and specific drug resistance profiles in different types of leukemia. Disclosure of Potential Conflicts of Interest Particularly interesting is the finding that a fraction of CML patients displays methylation of the ABCG2 promoter No potential conflicts of interest were disclosed. þ in CD34 progenitor cells, suggesting that these patients Acknowledgments may better respond to chemotherapeutic drugs. Overall, our findings suggest that deregulation of a We thank D. Manzoni for excellent work with cell cultures. specific set of ABC transporters may play a crucial role in dictating a drug resistance phenotype and tumor malig- Grant Support nancy in CML with numerous implications for CML treatment. Firstly, increased expression of ABC drug trans- This work was supported by The Italian Association for Research on Cancer porters by the BCR-ABL/c-MYC network, particularly in (AIRC, Italy), The Italian Ministry of University and Research (Italy) and University of Bologna (Italy), The National Health and Medical Research the cancer stem cell population, may explain for their self- Council, Australia (M.D. Norris, M. Haber) and the Cancer Institute New South renewal potential and for less sensitivity to drug treatments. Wales, Australia (M.D. Norris, M. Haber) Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and This last observation is consistent with a recent view on how Sydney Children's Hospital. drug resistance can be mediated by stem cells. According to The costs of publication of this article were defrayed in part by the payment this model, tumors contain a small population of cancer of page charges. This article must therefore be hereby marked advertisement in stem cells that produce differentiated offspring that are accordance with 18 U.S.C. Section 1734 solely to indicate this fact. committed to a particular lineage. Following chemother- Received November 11, 2010; revised June 9, 2011; accepted June 14, apy, the committed cells are killed, but the stem cells, 2011; published OnlineFirst June 21, 2011.

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c-MYC Oncoprotein Dictates Transcriptional Profiles of ATP-Binding Cassette Transporter Genes in Chronic Myelogenous Leukemia CD34+ Hematopoietic Progenitor Cells

Antonio Porro, Nunzio Iraci, Simona Soverini, et al.

Mol Cancer Res 2011;9:1054-1066. Published OnlineFirst June 21, 2011.

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