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Mir-139-5P Controls Translation in Myeloid Leukemia Through EIF4G2

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Mir-139-5P Controls Translation in Myeloid Leukemia Through EIF4G2 Oncogene (2016) 35, 1822–1831 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE miR-139-5p controls translation in myeloid leukemia through EIF4G2 S Emmrich1,9, F Engeland1,9, M El-Khatib1, K Henke1, A Obulkasim2, J Schöning1, JE Katsman-Kuipers2, C Michel Zwaan2, A Pich3, J Stary4, A Baruchel5, V de Haas6, D Reinhardt7, M Fornerod2,8, MM van den Heuvel-Eibrink2 and JH Klusmann1 MicroRNAs (miRNAs) are crucial components of homeostatic and developmental gene regulation. In turn, dysregulation of miRNA expression is a common feature of different types of cancer, which can be harnessed therapeutically. Here we identify miR-139-5p suppression across several cytogenetically defined acute myeloid leukemia (AML) subgroups. The promoter of mir-139 was transcriptionally silenced and could be reactivated by histone deacetylase inhibitors in a dose-dependent manner. Restoration of mir-139 expression in cell lines representing the major AML subgroups (t[8;21], inv[16], mixed lineage leukemia-rearranged and complex karyotype AML) caused cell cycle arrest and apoptosis in vitro and in xenograft mouse models in vivo. During normal hematopoiesis, mir-139 is exclusively expressed in terminally differentiated neutrophils and macrophages. Ectopic expression of mir-139 repressed proliferation of normal CD34+-hematopoietic stem and progenitor cells and perturbed myelomonocytic in vitro differentiation. Mechanistically, mir-139 exerts its effects by repressing the translation initiation factor EIF4G2, thereby reducing overall protein synthesis while specifically inducing the translation of cell cycle inhibitor p27Kip1. Knockdown of EIF4G2 recapitulated the effects of mir-139, whereas restoring EIF4G2 expression rescued the mir-139 phenotype. Moreover, elevated miR-139-5p expression is associated with a favorable outcome in a cohort of 165 pediatric patients with AML. Thus, mir-139 acts as a global tumor suppressor-miR in AML by controlling protein translation. As AML cells are dependent on high protein synthesis rates controlling the expression of mir-139 constitutes a novel path for the treatment of AML. Oncogene (2016) 35, 1822–1831; doi:10.1038/onc.2015.247; published online 13 July 2015 INTRODUCTION of mir-139 is associated with a favorable prognosis in hepatocellular Hematopoietic stem and progenitor cells (HSPCs), which acquire carcinoma and deletion of DLEU2/mir-15a/16-1 cluster, a gate- mutations conferring a maturation arrest and clonal proliferation, keeper of B-cell proliferation, leads to chronic lymphocytic represent the origin of acute myeloid leukemia (AML).1 Over the leukemia.9,10 In AML, downregulation of mir-181 indicated poor last decades, improved understanding of the underlying mole- prognosis in cytogenetically abnormal karyotypes.11 cular basis and the characterization of cytogenetically defined Therefore, it is not surprising that several recent reports subgroups led to the development of effective therapeutic demonstrated deregulation of mRNA translation to promote 12,13 strategies and to an improved outcome.2 Taking this into account, cellular transformation and a malignant phenotype. Translation the new World Health Organization classification scheme for regulation is primarily achieved during initiation, which requires myeloid neoplasms classifies the disease according to cytogenetic the protein complex known as eukaryotic initiation factor 4 F criteria including the chromosomal translocations t(8;21)(q22;q22) (EIF4F), consisting of three proteins: cap-binding protein EIF4E, (RUNX1-RUNX1T1), inv(16)(p13q22) (CBF-MYH11), t(15;17)(q22; scaffolding protein EIF4G and ATP-dependent RNA helicase q21) (PML-RARα), rearrangements of the mixed lineage leukemia EIF4A.14 The aberrant expression of EIF4F components, such as – (MLL) gene, and molecular changes in WT1, NPM1, CEBPA and EIF4E and EIF4G, has been linked to leukemia progression.15 17 DNMT3A.2,3 A series of recent studies has demonstrated the Therefore, targeting translation initiation emerged as a new importance of microRNAs (miRNAs) for blood homeostasis and the paradigm for AML therapy.18 This can be achieved by disrupting emergence of pediatric AML.4–6 MiRNAs are posttranscriptional the initiation complex using the 4EGI-1 compound, anti-EIF4E regulators, which control gene expression by modifying the antisense oligonucleotides, the antiviral drug ribavirin or by mRNA stability or translational initiation/elongation.7 Deregulation second-generation mammalian target of rapamycin inhibitors of miRNA-expression has been shown for solid tumors and (TORKinibs). In mammalian cells, the eukaryotic translation hematopoietic malignancies, indicating the importance of post- initiation factor 4, gamma (EIF4G) is present as three homologs, transcriptional deregulation for malignant transformation or EIF4G1, EIF4G2 (alias: death associated protein 5, DAP5; p97) and maintenance of malignant growth.8 For example, overexpression EIF4G3.19–21 The homologs are biochemically similar, but have 1Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany; 2Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands; 3Institute for Toxicology, Hannover Medical School, Hannover, Germany; 4Department of Pediatric Hematology/Oncology, Charles University and University Hospital Motol, Prague, Czech Republic; 5Department of Hematology, Hopitaux universitaires Saint-Louis, St Louis Hospital, Paris, France; 6Dutch Childhood Oncology Group (DCOG), Stichting Kinderoncologie Nederland (SKION), Hague, The Netherlands; 7Clinic for Pediatrics III, University Hospital Essen, Essen, Germany and 8Department of Biochemistry, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands. Correspondence: Dr JH Klusmann, Department of Pediatric Hematology and Oncology, Hannover Medical School, Carl-Neuberg-Street 1, Hannover D-30625, Germany. E-mail: [email protected] 9These authors equally contributed to this work. Received 5 December 2014; revised 21 May 2015; accepted 22 May 2015; published online 13 July 2015 mir-139 controls translation S Emmrich et al 1823 overlapping as well as divergent functional roles.21,22 Although In conclusion, mir-139 reduces proliferation, colony formation, elevated levels of EIF4G1 were shown to correlate with a cell viability and cell cycle activity in a broad range of AML cell malignant cell transformation,15,16,23 little is known about EIF4G2 lines representing the major AML subgroups. expression and its function in translation initiation. Here we describe the function of the epigenetically regulated Restoration of mir-139 expression has an anti-leukemic effect microRNA-139-5p that suppresses leukemic growth in AML cell in vivo lines in vitro and in vivo by targeting EIF4G2. By disrupting the To evaluate the anti-leukemic effect of mir-139 in vivo,we EIF4F initiation complex, mir-139 interferes globally with transla- performed xenotransplantation experiments using KASUMI-1 cells tional initiation and thereby reduces overall protein synthesis. with (8;21). As the t(8;21) cell context tolerates the highest amount of endogenous miR-139-5p (Figure 1a), using this cell line would RESULTS exclude artificial effects introduced by ectopic miRNA expression in an aberrant cellular context. In a competitive transplantation MiR-139-5p is suppressed in pediatric AML assay, NRG mice were intrafemorally (i.f.) transplanted with mir- fi Our recent miRNA expression pro ling in a cohort of pediatric AML 139-transduced (GFP+) or miR-NSC(ctrl)-transduced (mCherry+) cases followed by a gain-of-function screening indicated a putative 4 cells mixed together in a ratio of 1:1 (Figure 2a). The ratio shifted role of mir-139 as miRNA tumor suppressor. MiR-139-5p was to 1:17 (GFP+:mCherry+) in the bone marrow of the recipients after highly expressed in human terminally differentiated, post-mitotic 28 days, pointing toward a strong growth disadvantage conferred macrophages and neutrophils (22% and 11% of RNU48, respec- – by mir-139. In a second competitive transplantation setting, mice tively), but was low to absent in pediatric AML (0.01 0.24% of were transplanted with mir-139-transduced (GFP+) or miR-NSC RNU48) and normal HSPCs (0.18% of RNU48; Figure 1a). (ctrl)-transduced (GFP+) cells mixed together in a ratio of 1:1. A similar expression pattern can be found in normal mouse Quantification of the genomic copy number of vector-derived hematopoietic cells (Supplementary Figure 1a). In patients with miR-NSC- or mir-139 expression cassettes by quantitative PCR MLL-rearrangements, t(7;12), t(15;17) or normal karyotype miR-139- 27 days post transplantation again showed a 17.5-fold shift of the 5p was mainly absent. In cases with the t(8;21) and inv(16) AML mir-139:miR-NSC ratio at the favor of miR-NSC (Figure 2b; for (generally referred to as core-binding factor leukemias, CBF AML) as + quantitative PCR validation, see Supplementary Figures 2a and b). well as in normal CD34 -HSPCs, low levels of miR-139-5p were In a third assay, mice were intrafemorally transplanted with either detected (Figure 1a). CBF AMLs generally represent an AML 24 miR-NSC (ctrl) or mir-139-transduced KASUMI-1 cells and sub- subgroup with a favorable outcome. Comparing the expression jected to survival analysis (Figure 2c). mir-139 conferred a survival levels of both mature miRNAs strands confirmed miR-139-5p to be benefit to the transplanted mice (PMantel–Cox = 0.022) with an
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