Fusion Proteins MLL . AF4 and AF4 . MLL Confer Resistance to Apoptosis, Cell Cycling Capacity and Growth Transformation
Total Page：16
File Type：pdf, Size：1020Kb
Oncogene (2007) 26, 3352–3363 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Combined effects of the two reciprocal t(4;11)fusion proteins MLL . AF4 and AF4 . MLL confer resistance to apoptosis, cell cycling capacity and growth transformation A Gaussmann1, T Wenger2,3, I Eberle1, A Bursen1, S Bracharz1, I Herr2, T Dingermann1 and R Marschalek1 1Institute of Pharmaceutical Biology/ZAFES, JWG-University Frankfurt, Biocenter, Frankfurt/Main, Germany and 2Molecular OncoSurgery, Department of Surgery, University of Heidelberg and German Cancer Research Center, Heidelberg, Germany The reciprocal chromosomal translocation t(4;11)is Introduction correlated with infant, childhood, adult and therapy- related high-risk acute leukemia. Here, we investigated Chromosomal translocations t(4;11) are the most the biological effects of MLL . AF4, AF4 . MLL or the frequent chromosomal aberration of the MLL gene combination of both reciprocal fusion proteins in a and are correlated with infant, childhood, adult and conditional in vitro cell culture model system. Several therapy-related acute lymphoblastic leukemia (Felix parameters like cell growth, cell cycling capacity, et al., 1995; for a review see Mitterbauer-Hohendanner apoptotic behavior and growth transformation were and Mannhalter, 2004). In particular, t(4;11) patients investigated under physiological and stress conditions. have a poor prednisone response (Dordelmann et al., Co-transfected cells displayed the highest resistance 1999) and are therefore stratified according to high-risk against apoptotic triggers, cell cycling capacity and loss- acute leukemia protocols (Pui et al., 1994). of-contact inhibition. These analyses were complemented Although t(4;11) translocations are associated with by gene expression profiling experiments and specific gene such a severe disease phenotype, no animal model is signatures were established for each of the three cell lines. currently available that phenocopies the human disease. Interestingly, co-transfected cells strongly upregulate the By using the inverter mouse model and an AF4 knock-in homeobox gene Nanog. In combination with Oct4, the strategy, however, two different transgenic MLL . AF4 Nanog homeoprotein is steering maintenance of pluripo- mouse models have been established recently. Both tency and self-renewal in embryonic stem cells. Transcrip- developed a disseminated large B-cell lymphoma with tion of Nanog and other stem cell factors, like Oct4 and low penetrance and long latency: about 50% of these Bmi1, was verified in biopsy material of t(4;11)patient mice died of B-cell lymphomas after a median time of cells which express both reciprocal t(4;11)fusion genes. In 520–540 days (Chen et al., 2006; Metzler et al., 2006). conclusion, the presence of both reciprocal MLL fusion This clearly indicated that the MLL . AF4 fusion protein proteins confers biological properties known from t(4;11) alone does not cause the known t(4;11) disease leukemia, suggesting that each of the two fusion proteins phenotype. Besides these transgenic attempts, retroviral contribute specific properties and, in combination, also transduction of the MLL . AF4 fusion gene does not lead synergistic effects to the leukemic phenotype. to the development of any hematomalignancy (So et al., Oncogene (2007) 26, 3352–3363. doi:10.1038/sj.onc.1210125; 2004; Lavau et al., 2004, personal communication), even published online 27 November 2006 though a small number of blast-like replating cells could be obtained in semisolid agar. Keywords: MLL; AF4; acute leukemia; t(4;11) fusion Apart from these animal model systems, only a few proteins; Nanog stable cell lines were established from t(4;11) patients; however, they all differ in their biological properties and their karyotypes (Stong et al., 1985; Lange et al., 1987; Greil et al., 1994). They are characterized by the expression of lymphatic and myeloid surface antigens (e.g. CD34 þ , CD19 þ , CD13 þ , CD33 þ , CD133 þ , CD10À) and their ability to efficiently blockapoptosis Correspondence: Professor R Marschalek, Institute of Pharmaceutical under cytotoxic drug treatment (Kersey et al., 1998). Biology/ZAFES, University of Frankfurt, Biocenter, Max-von-Laue- This biological feature may explain the high relapse rate Str. 9, D-60439 Frankfurt/Main, Germany. in these leukemia patients. By contrast, overexpression E-mail: [email protected] of the MLL . AF4 fusion protein in the myelomonocytic 3Present address: Centre d’Immunologie de Marseille-Luminy, Mar- seille, France. leukemia cell line U937 cells caused cell cycle arrest Received 11 July 2006; revised 2 October 2006; accepted 13 October (Caslini et al., 2004), whereas siRNA-mediated knock- 2006; published online 27 November 2006 down of the MLL . AF4 fusion protein in the t(4;11) t(4;11) pathobiology A Gaussmann et al 3353 leukemic cell line SEM led to a strong increase of apoptosis and a reduction in clonogenicity (Thomas et al., 2005). Overexpression of the reciprocal AF4 . MLL fusion protein in MEF led to a loss-of- contact inhibition, and thus to growth transformation (Bursen et al., 2004). All these data sound controversial and did not permit consistent conclusions to be drawn on the pathological disease mechanism(s) of t(4;11) leukemia. Therefore, we aimed to establish an in vitro t(4;11) model system that recapitulates certain aspects of Figure 1 Doxycycline-dependent expression of MLL fusion genes t(4;11) leukemia cells, and thus allows investigation of and protein expression in stably transfected cells. (a) RT–PCR experiments. M ¼ size marker; A ¼ AF4 . MLL-transfected cell line; the specific contribution(s) of each reciprocal MLL B ¼ MLL . AF4-transfected cell line; C ¼ co-transfected cell line; fusion protein. N ¼ negative (water) control; P ¼ positive control (cloned cDNA); For this purpose, mammalian cells were stably 7Dox ¼ presence or absence of 10 mg/ml doxycycline. (b) Western transfected with conditional expression constructs cod- blot experiments. Lane 1: untransfected MEF/tTA cell line; lanes . 2–5: MEF/tTA cell lines stably transfected with Tet-off expression ing for doxycycline-inducible MLL AF4 and/or constructs coding for AF4 . MLL (der4), MLL . AF4 (der11) or AF4 . MLL transgenes. After induction, transfected cells both (der4/11); protein expression was induced by omitting were analysed for their growth properties (cell counts doxycycline in the growth media for 5 days. Upper panel: and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo- expression of the AF4 . MLL fusion protein; middle panel: lium bromide (MTT) assay), cell cycling capacity, expression of the MLL . AF4 fusion protein; lower panel: b-actin loading controls. Transfected cell lines were treated with MG132 to specific apoptotic rates and growth transformation. In stabilize the expression of the full-length AF4 . MLL fusion protein parallel, gene expression profiling experiments were (328 kDa) that otherwise would be degraded via the proteasomal performed to gain insights into up- and downregulated pathways due to its ability to interact with the E3-ligases SIAH1 or genes due to the presence of either one or both SIAH2, respectively (Bursen et al., 2004). reciprocal MLL fusion proteins. The results obtained suggest that both reciprocal MLL fusion proteins are contributing to the disease phenotype of t(4;11) leuke- mic cells. Moreover, gene expression profiling experi- expression of MLL . AF4 and AF4 . MLL in all three ments revealed the transcriptional upregulation of clones used throughout this study. Nanog and Oct4 in co-transfected cells, whereas single- transfected cells did not show any upregulation of these genes. This novel feature was verified in the biopsy Cell growth, specific apoptosis and cell cycle analysis of material of t(4;11) patients. Nanog codes for an single- and co-transfected cells under normal conditions Antennapedia-class homeobox protein which is – in and serum deprivation conjunction with Oct4 and Sox2 – necessary and All cell clones were grown in the absence of doxycycline sufficient for the self-renewal and maintenance of for 5 days and investigated under normal cell culture embryonic stem cells (Chambers et al., 2003; Mitsui conditions (Dulbecco’s modified Eagle’s medium et al., 2003). The consequences of these novel findings (DMEM)/10%fetal calf serum (FCS)/1% Pen-Strep). will be discussed. A significantly higher growth rate was observed for co- transfected cells, followed by untransfected MEF/tTA cells and AF4 . MLL-transfected cells. Nearly no growth Results was observed for MLL . AF4-transfected cells, indicating that these cells are inhibited in their growth potential Establishing conditional cell lines expressing MLL . AF4, (Figure 2a, see below). Noteworthy, AF4 . MLL-trans- AF4 . MLL or both MLL fusion genes fected and co-transfected cells displayed significantly Stably transfected MEF/tTA cell lines (Tet-off) were higher growth rates according to the data obtained by generated by transfecting them with the above men- the MTT assay, but the net growth of AF4 . MLL- tioned expression constructs. Single-cell clones were transfected cells seems to be compromised due to a selected under appropriate conditions and tested for higher apoptosis rate (2–20% over the observation their ability to induce their transgene(s) (data not period; see below). All other cell lines displayed an shown). Inducible cell clones were chosen and used apoptosis rate below 3% under these cell culture throughout the experiments. As shown in Figure 1a, a conditions (Figure 2b). The MTT assays revealed that doxycycline-dependent increase in transcription could the MEF/tTAHder11 cells displayed significantly lower be demonstrated for all transgenes after withdrawal of growth rates than the MEF/tTAHder4 and the MEF/ doxycycline (À). Time course experiments revealed tTAHder4/11 cell lines, respectively. It was also obvious expression of the resulting fusion proteins after 3–5 that untransfected control cells and MLL . AF4-trans- days (data not shown). Therefore, all subsequent fected cells seem to reduce their growth rates when experiments were performed after 5 days of transgene confluency was reached, whereas AF4 .