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 Williams 1 1 Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London; 2 Division of Molecular Immunology, National Institute for Medical Research, London, United Kingdom; and 3 Department of Child Health, Queen’s University, Belfast, United Kingdom Abstract The t[(11;19)(p22;q23)] translocation, which gives rise to the MLL-ENL fusion protein, is commonly found in infant acute leukemias of both the myeloid and lymphoid lineage. To investigate the molecular mechanism of immortalization by MLL-ENL we established a Tet-regulatable system of MLL- ENL expression in primary hematopoietic progenitor cells. Immortalized myeloid cell lines were generated, which are dependent on continued MLL-ENL expression for their survival and proliferation. These cells either terminally differentiate or die when MLL-ENL expression is turned off with doxycycline. The expression profile of all 39 murine Hox genes was analyzed in these cells by real-time quantitative PCR. This analysis showed that loss of MLL- ENL was accompanied by a reduction in the expression of multiple Hoxa genes. By comparing these changes with Hox gene expression in cells induced to differentiate with granulocyte colony-stimulating factor, we show for the first time that reduced Hox gene expression is specific to loss of MLL-ENL and is not a consequence of differentiation. Our data also suggest that the Hox cofactor Meis-2 can substitute for Meis-1 function. Thus, MLL-ENL is required to initiate and maintain immortalization of myeloid progenitors and may contribute to leukemogenesis by aberrantly sustaining the expression of a ‘‘Hox code’’ consisting of Hoxa4 to Hoxa11 . (Cancer Res 2005; 65(20): 9245-52) Introduction Translocations involving the mixed lineage leukemia (MLL ) gene on chromosome band 11q23 are associated with leukemias of both the myeloid and lymphoid lineage (1, 2). MLL translocations are most prevalent in infant leukemia where they comprise 80% of cases of acute lymphoblastic leukemia and 60% of cases of acute myeloid leukemia (AML; ref. 3). Infant leukemias bearing MLL translocations tend to have a particularly poor prognosis (3). MLL is the human homologue of the Drosophila trithorax (TRX ) gene (4). Gene targeting studies in mice have revealed that MLL is essential for definitive hematopoiesis and that it is required to maintain, but not to initiate, the expression of multiple Hox genes during embryogenesis (5–9). Some Hox genes are oncogenic when overexpressed in hematopoietic progenitor cells (10, 11). Taken together, this suggests that aberrant regulation of Hox genes by MLL fusion proteins is the basis for leukemias involving MLL translocations (12). Several recent publications do indeed suggest that Hox genes may play an important role in leukemia induced by MLL fusion proteins (13–16). MLL translocations result in the generation of an in-frame chimeric fusion in which MLL is joined to one of over 40 different partner genes, of which the most common are ENL, AF9 , and AF4 (17). The t[(11;19)(p22;q23)] translocation results in the fusion of the MLL gene to the eleven-nineteen-leukemia (ENL ) gene. Different murine models have been generated which recapitulate MLL-ENL–mediated leukemia. Immortalized myeloid and B-cell lines, both capable of inducing leukemia in vivo , have been generated by retroviral transduction of hematopoietic progenitor cells with MLL-ENL (18, 19). An interchromosomal recombination model has also been developed in which the de novo translocation of the MLL and ENL loci occurred specifically in hematopoietic cells (20). These mice developed AML with high penetrance and short latency, suggesting that the MLL-ENL translocation is the only event required for the development of leukemia (20). To gain more insight into the molecular mechanism of 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 with the Tet-Off conditional expression system to regulate the expression of the full-length MLL-ENL fusion protein. We determined whether continued MLL-ENL expression was required to maintain as well as initiate myeloid immortalization, and analyzed the expression profile of all 39 murine Hox genes in MLL- ENL immortalized cell lines. Materials and Methods Mice. All mice were maintained in the animal facilities of the National 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 constructed by subcloning the flag-tagged 5VMLL cDNA fragment (amino acids 1-1251; kindly provided by A. Biondi; ref. 21) into a modified version of pMSCV-neo (BD Clontech, Palo Alto, CA) upstream of the phosphoglycerate kinase (PGK) promoter and neo r . The 3V MLL-ENL cDNA (amino acids 1,252- 1,444 of MLL and amino acids 5-559 of ENL; kindly provided by D.C. Tkachuk; ref. 22) was then ligated in-frame downstream of the 5V MLL cDNA Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Owen Williams, Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, United Kingdom. Phone: 44-20-7813-8192; Fax: 44-20-7813-8100; E-mail: owen.williams@ich.ucl.ac.uk. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-1691 www.aacrjournals.org 9245 Cancer Res 2005; 65: (20). October 15, 2005 Research Article Research. on February 16, 2016. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from