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Continuous MLL-ENL Expression Is Necessary to Establish a ''Hox Code'' and Maintain Immortalization of Hematopoietic Progenitor Cells

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Continuous MLL-ENL Expression Is Necessary to Establish a ''Hox Code'' and Maintain Immortalization of Hematopoietic Progenitor Cells Research Article Continuous MLL-ENL Expression Is Necessary to Establish a ‘‘Hox Code’’ and Maintain Immortalization of Hematopoietic Progenitor Cells Sarah J. Horton,1 David G. Grier,3 Glenda J. McGonigle,3 Alexander Thompson,3 Michelle Morrow,1 Inusha De Silva,1 Dale A. Moulding,1 Dimitris Kioussis,2 Terence R.J. Lappin,3 Hugh J.M. Brady,1 and Owen Williams1 1Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London; 2Division of Molecular Immunology, National Institute for Medical Research, London, United Kingdom; and 3Department of Child Health, Queen’s University, Belfast, United Kingdom Abstract essential for definitive hematopoiesis and that it is required to The t[(11;19)(p22;q23)] translocation, which gives rise to the maintain, but not to initiate, the expression of multiple Hox genes MLL-ENL fusion protein, is commonly found in infant acute during embryogenesis (5–9). Some Hox genes are oncogenic when leukemias of both the myeloid and lymphoid lineage. To overexpressed in hematopoietic progenitor cells (10, 11). Taken investigate the molecular mechanism of immortalization by together, this suggests that aberrant regulation of Hox genes by MLL-ENL we established a Tet-regulatable system of MLL- MLL fusion proteins is the basis for leukemias involving MLL ENL expression in primary hematopoietic progenitor cells. translocations (12). Several recent publications do indeed suggest Immortalized myeloid cell lines were generated, which that Hox genes may play an important role in leukemia induced by are dependent on continued MLL-ENL expression for their MLL fusion proteins (13–16). survival and proliferation. These cells either terminally MLL translocations result in the generation of an in-frame differentiate or die when MLL-ENL expression is turned chimeric fusion in which MLL is joined to one of over 40 different off with doxycycline. The expression profile of all 39 murine partner genes, of which the most common are ENL, AF9, and AF4 Hox genes was analyzed in these cells by real-time (17). The t[(11;19)(p22;q23)] translocation results in the fusion of the MLL gene to the eleven-nineteen-leukemia (ENL)gene. quantitative PCR. This analysis showed that loss of MLL- ENL was accompanied by a reduction in the expression of Different murine models have been generated which recapitulate multiple Hoxa genes. By comparing these changes with Hox MLL-ENL–mediated leukemia. Immortalized myeloid and B-cell gene expression in cells induced to differentiate with lines, both capable of inducing leukemia in vivo, have been granulocyte colony-stimulating factor, we show for the first generated by retroviral transduction of hematopoietic progenitor time that reduced Hox gene expression is specific to loss of cells with MLL-ENL (18, 19). An interchromosomal recombination MLL-ENL and is not a consequence of differentiation. Our model has also been developed in which the de novo translocation data also suggest that the Hox cofactor Meis-2 can substitute of the MLL and ENL loci occurred specifically in hematopoietic for Meis-1 function. Thus, MLL-ENL is required to initiate cells (20). These mice developed AML with high penetrance and and maintain immortalization of myeloid progenitors and short latency, suggesting that the MLL-ENL translocation is the only event required for the development of leukemia (20). may contribute to leukemogenesis by aberrantly sustaining the expression of a ‘‘Hox code’’ consisting of Hoxa4 to To gain more insight into the molecular mechanism of Hoxa11. (Cancer Res 2005; 65(20): 9245-52) immortalization by the MLL-ENL fusion protein, we established a conditional system for MLL-ENL expression in murine hematopoi- etic progenitor cells. We used retroviral delivery in combination Introduction with the Tet-Off conditional expression system to regulate the Translocations involving the mixed lineage leukemia (MLL) gene expression of the full-length MLL-ENL fusion protein. We on chromosome band 11q23 are associated with leukemias of both determined whether continued MLL-ENL expression was required the myeloid and lymphoid lineage (1, 2). MLL translocations are to maintain as well as initiate myeloid immortalization, and most prevalent in infant leukemia where they comprise 80% of analyzed the expression profile of all 39 murine Hox genes in MLL- cases of acute lymphoblastic leukemia and 60% of cases of acute ENL immortalized cell lines. myeloid leukemia (AML; ref. 3). Infant leukemias bearing MLL translocations tend to have a particularly poor prognosis (3). Materials and Methods MLL is the human homologue of the Drosophila trithorax (TRX) Mice. All mice were maintained in the animal facilities of the National gene (4). Gene targeting studies in mice have revealed that MLL is Institute for Medical Research and experiments done according to National Institute for Medical Research institutional guidelines and United Kingdom Home Office regulations. Retroviral constructs. The pMSCV-MLL-ENL (MSCV-M/E) vector was Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). constructed by subcloning the flag-tagged 5VMLL cDNA fragment (amino Requests for reprints: Owen Williams, Molecular Haematology and Cancer acids 1-1251; kindly provided by A. Biondi; ref. 21) into a modified version of Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, pMSCV-neo (BD Clontech, Palo Alto, CA) upstream of the phosphoglycerate London WC1N 1EH, United Kingdom. Phone: 44-20-7813-8192; Fax: 44-20-7813-8100; kinase (PGK) promoter and neor. The 3VMLL-ENL cDNA (amino acids 1,252- E-mail: [email protected]. I2005 American Association for Cancer Research. 1,444 of MLL and amino acids 5-559 of ENL; kindly provided by D.C. doi:10.1158/0008-5472.CAN-05-1691 Tkachuk; ref. 22) was then ligated in-frame downstream of the 5VMLL cDNA www.aacrjournals.org 9245 Cancer Res 2005; 65: (20). October 15, 2005 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Research fragment. For some experiments, the flag tag was replaced with a Myc tag. Real-time quantitative PCR. Quantitative PCR reactions, using TaqMan The tetracycline transactivator (tTA) cDNA (BD Clontech) was subcloned probe based chemistry (Applera, Foster City, CA), were carried out and into pMSCV-internal ribosome entry site (IRES)-enhanced green fluorescent murine target amplicons were analyzed in a similar manner to the human protein (EGFP) upstream of the IRES-EGFP cassette to generate the MSCV- set, as previously described (24). All oligonucleotides were designed against tTA vector (23). The MSCV-tetracycline response element (TRE) vector was GenBank published sequences (Supplementary Table) using Primer Express generated by ligating the TRE (BD Clontech) downstream of the PGK-Neor software (Applera). Nucleotide sequences for oligonucleotide primers and in pMSCV-neo. The myc-tagged MLL-ENL cDNA fragment was then ligated probes are available on request. Total RNAs isolated from all major adult into MSCV-TRE downstream of the TRE to generate the MSCV-TRE-M/E and fetal tissues, including whole embryos, were pooled and used for the vector. validation of the murine Hox TaqMan primers and probes. The quantitative Isolation and infection of hematopoietic progenitor cells. Retroviral PCR analysis of Hox cofactor (Fig. 6) and FLT3 (Supplementary Fig. S4) gene supernatants were produced as described previously (23). Hematopoietic expression was done using predesigned TaqMan primers and probes progenitor cells were purified from single-cell suspensions of bone marrow, (Applera) and an ABI prism 7000 sequence detection system (Applera). extracted from C57BL/10 or C57BL/6 mice 5 days after i.v. injection of Standard curve generation. Amplicons generated using the forward 150 mg/kg 5-fluorouracil (Faulding Pharmaceuticals, Leamington Spa, and reverse primer pairs of the original Hox targets were cloned into United Kingdom). Magnetic activated cell sorting was used to purify c-Kit+ pCR2.1-TOPO TA (Invitrogen) or pGEM-T Easy (Promega, Southampton, hematopoietic progenitor cells using monoclonal antibody (mAb) specific to United Kingdom) using standard protocols. Plasmid DNA was prepared c-Kit (2B8; BD Biosciences, PharMingen, San Diego, CA), and lineage using the Qiagen miniprep kit (Qiagen Ltd., Crawley, United Kingdom) depleted hematopoietic progenitor cells, using mAbs from a lineage panel kit according to the instructions of the manufacturer. Quantitation of plasmid (BD PharMingen). Hematopoietic progenitor cells were cultured with 100 DNA was determined spectrophotometrically. An A260 value of 0.1 or higher ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3 (IL-3), and 10 ng/mL was deemed satisfactory and, in terms of plasmid purity, an A260/A280 of IL-6 (Peprotech EC, London, United Kingdom) for 24 hours before infection. 1.7 to 2.0 was accepted. The plasmid DNA concentration was converted to Hematopoietic progenitor cells were infected with retrovirus supplemented copy numbers of plasmid using the following formula: V (AL) = {1 Â 108 Â with the same growth factors on 2 consecutive days by spinoculation 309 Â [plasmid size + insert size (bp)]} / [plasmid concentration (Ag/AL) Â (centrifugation at 700 Â g,25jC, 45 minutes) in the presence of 5 Ag/mL 1 Â 10À6 Â 6.02 Â 1023]. Plasmids were diluted to 109 copies/AL and polybrene (Sigma-Aldrich, Poole, United Kingdom). Coinfections were done linearized with NotI (New England Biolabs, Beverly, MA). Quantitative PCR using unconcentrated MSCV-TRE-M/E and concentrated MSCV-tTA super- analyses were done in triplicate using serially diluted plasmids (in the natants. 101-107 copies/AL range) as templates, a primer and probe mixture, and Colony-forming assays and generation of myeloid cell lines. Universal Mastermix (Applied Biosystems). All standard curves, correlation Transduced hematopoietic progenitor cells were cultured in 1.1 mL coefficients, gradient, and intercept values were generated using the ABI duplicate methylcellulose cultures in 35 mm plates 24 hours after infection.
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