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. Ó2011 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 Ó2011 American Association for Cancer Research. of cancers, a drug-resistant phenotype has been linked to 1054 Mol Cancer Res; 9(8) August 2011 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst June 21, 2011; DOI: 10.1158/1541-7786.MCR-10-0510 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