Leukemia (2010) 24, 89–96 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu ORIGINAL ARTICLE Hsa-mir-125b-2 is highly expressed in childhood ETV6/RUNX1 (TEL/AML1) leukemias and confers survival advantage to growth inhibitory signals independent of p53 N Gefen1,2,8, V Binder1,3,8, M Zaliova4, Y Linka3, M Morrow6, A Novosel3, L Edry5, L Hertzberg1,2,7, N Shomron5, O Williams6, J Trka4, A Borkhardt3 and S Izraeli1,2 1Section of Functional Genomics and Leukemia Research, Department of Pediatric Hemato-Oncology, Sheba Medical Center, Tel-Hashomer, Israel; 2Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3Department of Pediatric Hematology, Oncology and Immunology, Heinrich Heine University, Duesseldorf, Germany; 4Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Childhood Leukemia Investigation Prague, Prague, Czech Republic; 5Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 6Institute of Child Health, University College London, London, UK and 7Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel MicroRNAs (miRNAs) regulate the expression of multiple characterized by an intrachromosomal amplification of a small proteins in a dose-dependent manner. We hypothesized that region within the long arm of chr 21 (iAMP21) around the increased expression of miRNAs encoded on chromosome 21 4 (chr 21) contribute to the leukemogenic function of trisomy 21. RUNX1 locus. In contrast to ETV6/RUNX1 and HHD ALL, it is The levels of chr 21 miRNAs were quantified by qRT–PCR in associated with poor prognosis. The most common structural four types of childhood acute lymphoblastic leukemia (ALL) chromosomal aberration in childhood ALL occurring in characterized by either numerical (trisomy or tetrasomy) or approximately 25% of pediatric B-cell precursor ALL fuses the structural abnormalities of chr 21. Suprisingly, high expression RUNX1 (AML1) gene on chr 21 with the ETV6 (TEL) gene on chr of the hsa-mir-125b-2 cluster, consisting of three miRNAs, 12.5,6 Although ETV6/RUNX1 causes a prenatal preleukemic was identified in leukemias with the structural ETV6/RUNX1 clonal expansion, additional genetic events are required for abnormality and not in ALLs with trisomy 21. Manipulation of 7,8 ETV6/RUNX1 expression and chromatin immunoprecipitation evolution of leukemia. studies showed that the high expression of the miRNA cluster MicroRNAs (miRNAs) are 20–25 nucleotide long non-coding is an event independent of the ETV6/RUNX1 fusion protein. RNAs that have a vital function in the regulation of protein Overexpression of hsa-mir-125b-2 conferred a survival advan- expression.9 Currently, there are six validated mature miRNAs tage to Ba/F3 cells after IL-3 withdrawal or a broad spectrum of encoded on human chr 21 (http://microrna.sanger.ac.uk apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the Figure 1a). Hsa-mir-99a, hsa-let-7c and hsa-mir-125b-2 are ETV6/RUNX1 leukemia cell line REH increased apoptosis after clustered together in the same intron of the C21ORF34 gene Doxorubicin and Staurosporine treatments. P53 protein levels on 21q.21.1 (NC_000021.7) (hsa-mir-125b-2 cluster). Hsa-mir- were not altered by miR-125b. Together, these results suggest 125b-1, the homolog of Caenorhabditis elegans lin-4, has been that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL shown to be expressed in solid tumors and associated with provides survival advantage to growth inhibitory signals in a enhanced cell proliferation and survival.10–12 An insertion of hsa- p53-independent manner. Leukemia (2010) 24, 89–96; doi:10.1038/leu.2009.208; mir-125b-1 into the IGH locus was described in a patient with B- published online 5 November 2009 cell precursor ALL suggesting its involvement in the leukemic 13 Keywords: acute lymphoblastic leukemia (ALL); ETV6/RUNX1; process. It has been shown to be expressed in myeloid TEL/AML1; microRNA malignancies and to block myeloid differentiation.14 Recently, it was also reported to protect from apoptosis through negative regulation of p53 in zebrafish, human neuroblastoma cells and Introduction lung fibroblasts, but not in mouse cells.15 Hsa-mir-155 has been linked to B-cell development. Others and we have shown its Aberrations in chromosome 21 (chr 21) are commonly found in increased expression in lymphomas.16–19 Strikingly, transgenic childhood acute lymphoblastic leukemia (ALL). The high- expression of miR-155 induced pre-B-cell leukemias and lymphomas 1,2 hyperdiploid ALL (HHD ALL), the most common type of in mice,20 thus miR-155 seems to be a potent oncogene. childhood ALL with numerical chromosomal aberrations, is The mechanisms of miRNA function suggest that their activity characterized by 3–4 copies of chr 21 and a variable presence of should be correlated with their dosage and that each miRNA other specific chromosomal trisomies. The markedly increased may regulate multiple targets.21 Thus, we hypothesized that 3 incidence of ALL in children with Down’s syndrome (DS) miRNAs contribute to the oncogenic effects of chromosomal strongly suggests that trisomy 21 is leukemogenic, but the chr 21 trisomies, a situation in which a small change ( Â 1.5) in genes involved in these leukemias are presently unknown. genomic dosage results in profound effects. This hypothesis is A newly discovered rare subtype of childhood ALL is strengthened by our recent observations showing a general increase in expression of multiple genes from the trisomic Correspondence: Professor S Izraeli, Section of Functional Genomics chromosomes.22 Thus, we expected that miRNAs from chr 21 and Leukemia Research, Department of Pediatric Hemato-Oncology, would be overexpressed in HHD and DS ALL (3–4 copies of chr Sheba Medical Center, Tel Hashomer, Ramat Gan 52621, Israel. 21) relative to ETV6/RUNX1 and iAMP21 leukemias. E-mail: [email protected] 8These two authors contributed equally to this work. Here, we report the surprising observations that unlike most of Received 2 February 2009; revised 17 August 2009; accepted 4 the genes encoded on chr 21, the expression of the hsa-mir- September 2009; published online 5 November 2009 125b-2 cluster does not correlate with gene copy number, but MicroRNA in ETV6/RUNX1 ALL N Gefen et al 90 miR-155 iAMP21- ETV6/RUNX1 ALL ALL 4.0 HHD ALL 3.5 DS ALL 3.0 iAMP21 ALL 2.5 p13 p11.2 q11.2 q21.2 q21.3 q22.11 q22.12 q22.13 q22.2 q22.3 q21.1 2.0 * * 1.5 1.0 * 0.5 0.0 miR-802 let-7c miR-99a miR-125b miR-155 “miR-125b cluster“: Hsa-miR-let-7c Hsa-miR-99a Hsa-miR-125b 3.5 ETV6/RUNX1 initial 3 ETV6/RUNX1 remission * 2.5 2 * * 1.5 1 * 0.5 0 let-7c miR-99a miR-125b miR-155 Figure 1 Expression of miRNAs encoded on chr 21 in different leukemia samples. (a) Schematic representation of the location of miRNAs encoded on chr 21. The level of the mature hsa-miR-802 could not be measured because of the lack of an appropriate qRT–PCR-based assay. (b, c) MiRNA expression was determined by qRT–PCR using the TaqMan MicroRNA Assays from Applied Biosystems. The numbers on the y axis represent relative expression levels of mature miRNA normalized to an internal control (RNU43). Standard errors are indicated. Asterisk marks significance. (b) Expression of the chr 21 miRNAs in diagnostic bone marrow samples of different ALL subtypes: ETV6/RUNX1 (24 patients), DS (10 patients), high hyperdiploid (10 patients), iAMP21 (7 patients) Po0.05 (ANOVA). (c) Comparison of miRNA expression between diagnosis and remission samples of ETV6/RUNX1 leukemias Po0.005 (T-test). rather is highly expressed in ETV6/RUNX1 ALL. Additional ALL (n ¼ 7). Surprisingly, extra copies of chr 21 did not research suggests that the expression of the hsa-miR-125b-2 contribute to the expression level of these miRNAs (Figure 1b). cluster is an independent event in ETV6/RUNX1 ALL, conferring Although hsa-mir-155 is similarly expressed between the survival advantage under growth inhibitory and apoptotic different ALLs, the other miRNAs hsa-mir-99a, hsa-let-7c and conditions in a p53-independent manner. hsa-mir-125b-2 are up-regulated in the ETV6/RUNX1 ALL, the subtype of leukemia with a structural aberration of chr 21. The expression was derived from the leukemic blasts, as it was Materials and methods not observed in remission samples (Figure 1c). To ensure that the observed results were not because of the normalization to a Patients single internal control (RNU 43), we have extended the miRNA RNA was derived from diagnostic or remission bone marrow profiling using TaqMan Low Density miRNA arrays (TLDA, ABI) samples of childhood ALL patients obtained with informed normalizing to two additional internal controls (RNU6 and consent. Diagnostic bone marrow samples contained at least RNU48). As can be seen in Supplementary Figure 1, the TLDA 80% lymphoblasts. The samples were anonymized before results are similar to the results obtained with the singleplex shipping except for the information on the genetic subgroup. miRNA expression profiling. The study was approved by the IRB of the Israeli Health Ministry and Sheba Medical Center. The rest of the material and methods are detailed in a Hsa-mir-125b-2 cluster is not a target of the ETV6/ supplementary file. RUNX1 fusion protein To test whether mir-125b-2 is a target of ETV6/RUNX1, we performed overexpression experiments in two mouse hemato- Results poietic progenitor cell systems: Ba/F3 cells and primary mouse embryonic fetal liver progenitors.