Leukemia (2010) 24, 601–612 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu ORIGINAL ARTICLE Functional characterization of high levels of meningioma 1 as collaborating oncogene in acute leukemia T Liu1, D Jankovic1, L Brault1, S Ehret1, F Baty1, V Stavropoulou1, V Rossi2, A Biondi2 and J Schwaller1 1Department of Biomedicine, University Hospital Basel, Basel, Switzerland and 2The Centro M. Tettamanti, Clinica Pediatrica, Universita` Milano-Bicocca, Monza, Italy Retroviral expression of leukemogenic oncogenes in the system by a conditional knock-in strategy resulted in the murine hematopoietic system is essential but not sufficient to formation of T-cell lymphomas as well as AML after a long induce acute leukemia. Proviral integration-mediated elevated latency, suggesting that MN1-TEL, similar to MLL-X fusions, is expression of the meningioma 1 (MN1) oncogene suggested 4–6 MN1 acting as cooperating event in mixed-lineage leukemia 1 essential but not sufficient to induce the disease. Gene (MLL) and eleven nineteen leukemia (ENL)-induced murine expression profiling studies of a large number of human leukemia leukemia. Indeed, co-expression of MN1 with MLL-ENL en- samples showed that MN1 is deregulated in cases with alterations hanced transformation in vivo, and resulted in a significantly at 3q26 leading to ecotropic virus integration-1 (EVI1) over- reduced latency for induction of an aggressive acute leukemia expression.7 In addition, elevated MN1 expression has been when compared with MN1 or MLL-ENL alone. In addition, co- associated with the presence of inv16 leading to a core-binding expression of MN1 increased the granulocyte macrophage 8 progenitor cell population with leukemia-initiating properties as factor-b/MYH11 fusion. Recently, functional studies have shown shown in secondary transplantation experiments. Gene expres- that overexpression of MN1 alone is able to induce an AML sion profiling experiments identified putative downstream phenotype in mice.8,9 Furthermore, high MN1 expression was MN1 targets, of which FMS-like tyrosine kinase 3 (FLT3) and shown to have negative prognostic effect in AML, especially in CD34 were upregulated in both MN1-overexpressing murine the absence of common karyotype abnormalities.10,11 leukemias and in pediatric acute leukemias with high MN1 levels. Interestingly, small interfering RNA (siRNA)-mediated Recurrent chromosomal translocations resulting in expression MN1 knockdown resulted in cell cycle arrest and impaired clono- of oncogenic fusion genes are the hallmark of human genic growth of human leukemia cell lines with high MN1 levels. leukemias.12 Mixed-lineage leukemia 1 (MLL1) on 11q23 Our work shows for the first time that high MN1 levels are is one of the most frequently altered genes in human leukemia important for the growth of leukemic cells, and that increased with MLL fusion genes involving over 50 partners or partial MN1 expression can synergize with MLL-ENL and probably tandem duplications.13,14 Several studies have shown that other transforming fusion genes in leukemia induction through a distinct gene expression program that is able to expand the a large number of MLL fusions as well as MLL-partial leukemia-initiating cell population. tandem duplications have in vitro and/or in vivo transforming Leukemia (2010) 24, 601–612; doi:10.1038/leu.2009.272; activity. Indeed, transplantation of bone marrow cells retro- published online 14 January 2010 virally expressing various MLL fusions (such as MLL-AF9, Keywords: meningioma1; acute leukemia; oncogene; collaboration MLL-ENL or MLL-GAS7) led to the induction of a lethal disease mimicking human acute leukemia. The clonal character as well as a latency period of 60 to 4200 days of the induced leukemia suggested that although MLL-X fusions are essential, they might Introduction not be fully sufficient for inducing a leukemic phenotype in vivo.15–19 The meningioma 1 (MN1) gene was first identified as the target Elevated MN1 expression was not only associated with the of a sporadic balanced chromosomal translocation in a patient presence of distinct genetic alterations (e.g. inv16, alterations of with meningioma.1 The absence of MN1 expression in the index the EVI1 gene locus) in human leukemia, but the MN1 gene patients has led to the suggestion that MN1 is a candidate tumor locus was also target of proviral integration site in mouse suppressor gene. Several studies have proposed that MN1 leukemia models induced by bone marrow reconstitution of retrovirally expressed oncogenes, such as mutant AML1 or presumably exerts an effect as a transcriptional cofactor, most 20,21 probably through interaction with other transcriptional regula- NUP98-HOXD13. Searching for potentially cooperating tors, such as p300/CREB-binding protein.2 MN1 was first linked events by cloning proviral integration sites in murine leukemia to human leukemia after the cloning of the balanced chromo- induced by retrovirally expressed MLL-ENL, we found increased somal translocation t(12;22)(p13;q12) in patients with acute MN1 expression in leukemic cells harboring an integration near myeloid leukemia (AML), myelodysplasia or chronic myelo- the MN1 locus. These observations suggested that elevated genous leukemia. This translocation leads to the expression of MN1 levels might synergize in leukemia induction initiated by an MN1–TEL fusion that consists of almost the entire open distinct genetic alterations such as MLL-ENL. By co-expression reading frame of MN1 fused with the DNA-binding moiety of of MN1 and MLL-ENL in the murine hematopoietic system, we TEL.3 Expression of MN1–TEL in the mouse hematopoietic were able to show that overexpression of MN1 can exert an effect as a collaborative genetic hit in MLL-ENL-induced leukemia in vivo through expansion of the pool of leukemia- Correspondence: Professor J Schwaller, Department of Biomedicine, initiating cells. New putative MN1 target genes were identified University Hospital Basel, Hebelstrasse 20, ZLF, Lab 318, Basel CH-4031, Switzerland. E-mail: [email protected] and validated in mouse and human leukemias overexpressing Received 25 May 2009; revised 2 November 2009; accepted 23 MN1. For the first time, we show that small interfering RNA November 2009; published online 14 January 2010 (siRNA)-mediated knockdown of MN1 impaired proliferation of Meningioma 1 as collaborating leukemogenic oncogene T Liu et al 602 human leukemic cells with abundant levels of MN1, suggesting days. Colonies were harvested, and 104 cells were replated in that MN1 could represent a new therapeutic target. methylcellulose for four rounds. Material and methods Analysis of transplanted mice After red cell lysis peripheral blood and bone marrow cells were Cell lines counted and analyzed using a flow cytometer (Cyan II, Becton The following human leukemia cell lines were analyzed: Dickinson, Franklin Lakes, NJ, USA), single-cell suspensions MV4;11, acute myeloid leukemia (MLL-AF4 þ , FMS-like were stained with phycoerythrin, or allophycocyanin fluoro- tyrosine kinase 3 (FLT3)-ITD þ ), MOLM13, acute myeloid chromes-labeled c-Kit, Sca1, Gr1, Mac1, B220 and CD34 leukemia (MLL-AF9 þ ); EOL1, acute eosinophilic leukemia monoclonal antibodies (all from Pharmingen, San Diego, CA, (MLL-partial tandem duplication); THP1, acute monocytic USA). Histopathological analysis of peripheral blood, bone leukemia (MLL-AF9 þ ); KOCL44, acute lymphoblastic leukemia marrow and hematopoietic organs were performed using (MLL-ENL þ ), SEM, acute lymphoblastic leukemia (MLL- standard procedures. AF4 þ ), KOPN8, acute lymphoblastic leukemia (MLL-ENL þ ), RS4;11, acute lymphoblastic leukemia (MLL-AF4 þ ) and HL-60, acute myeloid leukemia. All cells were kept in RPMI-1640 with Southern blot analysis Southern blot analysis was performed using standard protocols.22 Glutamine (Invitrogen, Carlsbad, CA, USA) plus 10% fetal Clonality of the MSCV-MLL-ENL provirus was assessed using a bovine serum and penicillin/streptomycin at 37 1C. 2.2 kb Hind III human MLL1 cDNA fragment as the hybridization probe.23 The Mn1 locus-specific probe was generated using PCR Construction of recombinant retroviral vectors and the primer sequences are available upon request. Full-length human 50-FLAG-tagged MN1 complementary DNA (cDNA) was excised (SacI-HindIII) from pCMVTag2B-MN1 (a kind Retroviral integration cloning by splinkerette PCR gift from Dr Paul MacDonald, Cleveland, OH) and transferred into Genomic DNA isolated from bone marrow or spleen cells of the pSL1180 (SacI-SmaI) cloning vector and further subcloned into leukemic mice was digested with NlaIII or MseI for 12–16 h, and pMSCV-IRES/EYFP.TheMLL-ENLcDNA(akindgiftfromDrRobert ligated to the splinkerette linker overnight. The nested PCR was Slany, Erlangen, Germany) was subcloned from pMSCV-pgk/neo performed by using splinkerette linker-specific primers and into pMSCV-IRES/EGFP (for single transductions) or pMSCV- primers recognizing the long terminal repeat of pMSCV.24,25 pgk/puro (for double transductions) using a unique EcoRI site. All Amplicons from the second PCR were separated on 2% agarose expression plasmids were verified by extensive restriction digests, gel, purified by gel purification kit (Qiagen, Hilden, Germany) and and by partial sequencing. subcloned into pCR 2.1-TOPO vector (Invitrogen) before sequen- cing, or directly sequenced by using the BigDye Terminator v3.1 chemistry and ABI 3130 DNA genetic analyzer (Applied Bone marrow infections and transplantation Biosystems, Foster City, CA, USA). The obtained sequences were Bone marrow cells were harvested from 6- to 10-week-old (FVB/ analyzed using BLAST against the mouse genome database of the N Â 129/S1)F1 mice 4 days after intraperitoneal injection of National Center for Biotechnology Information. 5-fluorouracil 150 mg/kg (Sigma, St Louis, MO, USA), and were cultured for 24 h in RPMI-1640 supplemented with 10% fetal bovine serum, 10 ng/ml of human interleukin-6 (IL-6), 6 ng/ml of siRNA knockdown experiments murine IL-3 and 100 ng/ml of murine stem cell factor The MN1-specific short hairpin RNA or scramble short hairpin (PeproTech EC, London, UK).