ORIGINAL ARTICLE Leukemic fusion genes MLL/AF4 and AML1/MTG8 support leukemic self-renewal by controlling expression of the telomerase subunit TERT A Gessner 1,8 , M Thomas 2,6,8 , P Garrido Castro 1 , L Bu ¨ chler 1 , A Scholz 1 , TH Bru ¨ mmendorf 3,4 , N Martinez Soria 2,7 , J Vormoor 1 , J Greil 5 and O Heidenreich 1 1 Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK; 2 Department of Molecular Biology, Interfaculty Institute for Cell Biology, Eberhard Karls University Tu ¨ bingen, Tu ¨ bingen, Germany; 3 Department of Hematology and Oncology, University Medical Center II, Tu ¨ bingen, Germany; 4 Department of Hematology and Oncology, University Hospital Aachen, Aachen, Germany and 5 Department of Hematology, Oncology, Immunology and Pneumology, Children’s Hospital, Heidelberg, Germany MLL/AF4 and AML/MTG8 represent two leukemic fusion genes, which are most frequently found in infant acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), respec- tively. We examined the influence of MLL/AF4 and AML1/MTG8 fusion genes on the expression of TERT coding for the telomerase protein subunit, and subsequently telomerase activity in t(4;11)-positive ALL and t(8;21)-positive cell lines, respectively. MLL/AF4 suppression diminished telomerase activity and expression of TERT. Blocking pro-apoptotic caspase activation in conjunction with MLL/AF4 knockdown enhanced the inhibition of TERT gene expression, which suggests that MLL/AF4 depletion does not reduce TERT expression levels by inducing apoptosis. Knockdown of HOXA7, a direct transcriptional target of MLL/AF4 fusion gene, caused a reduction of telomerase and TERT to an extent similar to that observed with MLL/AF4 suppression. Chromatin immunoprecipitation of SEM cells, using ectopically expressed FLAG-tagged Hoxa7, indicates HOXA7 binding site in the TERT promoter region. Furthermore, suppression of the AML1/MTG8 fusion gene was associated with severely reduced clonogeni- city, induction of replicative senescence, impaired TERT expression and accelerated telomere shortening. We thus present findings that show a mechanistic link between leukemic fusion proteins, essential for development and maintenance of leukemia, and telomerase, a key element of both normal and malignant self-renewal. Leukemia (2010) 24, 1751–1759; doi:10.1038/leu.2010.155; published online 5 August 2010 Keywords: self-renewal; telomerase; fusion gene; MLL/AF4; AML1/MTG8 Introduction The concept of tumorigenic leukemic stem cells (LSCs) emerged after a number of studies that found a subgroup of leukemic cells capable of extensive proliferation. 1 The hallmark of LSCs is their ability to self-renew and to generate and maintain the full spectrum of leukemic cell heterogeneity found in a given leukemia. On the basis of this principle, it was hypothesized that most current treatment methods target the descendent bulk of LSCs while leaving the LSC themselves unaffected. Indeed, there is ample evidence to suggest that LSC have a crucial role in therapy resistance and relapse. 2 To acknowledge this, novel treatment approaches targeting aberrant self-renewal of cancer stem cells, including LSCs, are being explored. However, our current understanding of the molecular mechanisms underlying malignant self-renewal is still incomplete. Thus, the development of novel therapies, designed to target the LSC niche, is only just emerging. Both normal and malignant self-renewal crucially depend on preserving telomeres to maintain genomic integrity and to prevent replicative senescence. 3 Telomere ends are restored by telomerase, a reverse transcriptase consisting of a protein subunit, TERT, and an RNA subunit, TERC (also termed hTR). TERC serves as a template for telomere synthesis and is ubiquitously expressed, whereas, in contrast, transcription of TERT is tightly regulated by transcription factors such as SP1, c-MYC and IRF-4, and is repressed by, for example, the transforming growth factor-b signal transduction pathway. 4–6 TERT activity is upregulated during cell-cycle activation of immature cells and expression is reduced during cell differ- entiation. Among the different components of the telomerase ribonucleoprotein complex, TERT is regarded as the rate- limiting component for telomerase activity. Leukemic fusion genes are generated by chromosomal translocations and are hallmarks of leukemia. Fusion genes, such as AML1/MTG8 (RUNX1/RUNX1T1 or AML1/ETO) or MLL/AF4 (MLL/AFF1 or ALL1/AF4), are exclusively expressed in preleukemic and leukemic cells. The AML1/MTG8 fusion gene is generated from a chromosomal translocation t(8;21) and represents the most common aberration associated with 10–15% of acute myeloid leukemia (AML). This translocation fuses AML1 (or RUNX1), a transcription factor for definitive hemopoiesis, with MTG8 (or RUNXT1), which is part of histone deacetylase-containing complexes. 7–9 Thus, it converts a transcriptional modulator to a repressor of gene expression by interfering with chromatin modification. In contrast, the chromosomal translocation t(4;11) fuses the MLL gene (mixed lineage leukemia) on chromosome 11 with the AF4 gene on chromosome 4 thereby generating two fusion genes, the derivative 4, AF4/MLL, and the derivative 11, MLL/AF4. 10–12 This translocation causes an aggressive type of acute lympho- blastic leukemia (ALL), which predominantly affects infants, and is associated with an unfavorable prognosis. 13 Both fusion genes can originate already prenatally. 14,15 They are key to maintaining a malignant phenotype, hence Received 23 April 2010; revised 8 June 2010; accepted 22 June 2010; published online 5 August 2010 Correspondence: Dr O Heidenreich, Northern Institute for Cancer Research, Newcastle University, Paul O’Gorman Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. E-mail: olaf.heidenreich@ncl.ac.uk 6 Current address: Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany. 7 Current address: Departamento de Biotecnologı ´a Microbiana, Centro Nacional de Biotecnologı ´a, Madrid, Spain. 8 These authors contributed equally to this work. Leukemia (2010) 24, 1751–1759 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu