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Cooperative Transformation by MEF2D/DAZAP1 and DAZAP1/MEF2D Fusion Proteins Generated by the Variant T (1; 19) in Acute Lymphoblastic Leukemia

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Cooperative Transformation by MEF2D/DAZAP1 and DAZAP1/MEF2D Fusion Proteins Generated by the Variant T (1; 19) in Acute Lymphoblastic Leukemia Leukemia (2007) 21, 2470–2475 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE Cooperative transformation by MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins generated by the variant t(1;19) in acute lymphoblastic leukemia V Prima1,2 and SP Hunger1,2 1Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA and 2University of Florida Shands Cancer Center, Gainesville, FL, USA A variant t(1;19)(q23;p13.3) translocation creates reciprocal DAZAP1/MEF2D and MEF2D/DAZAP1 fusion transcripts.4,5 DAZAP1/MEF2D and MEF2D/DAZAP1 fusion genes that are MEF2D is a member of the MEF2 family of DNA-binding expressed in acute lymphoblastic leukemia. We used retroviral gene transfer to ectopically express wild-type and chimeric proteins, which activate transcription of genes involved in DAZAP1 and MEF2D fusion proteins in NIH 3T3 cells. In soft control of differentiation and survival of lymphoid, neural and 6 agar assays, each of the fusion proteins transformed 3T3 cells muscle cells. DAZAP1 is a novel RNA-binding protein involved with a 20-fold increase in colony formation as compared in mRNA processing/trafficking that is expressed most abun- to empty vector or native MEF2D or DAZAP1 proteins. dantly in testis.7 Co-expression of both DAZAP1/MEF2D and MEF2D/DAZAP1 The reciprocal chimeric proteins created by this t(1;19) led to a threefold increase in colony formation as compared to either fusion protein alone. Expression of wild-type DAZAP1, include important functional domains of DAZAP1 and MEF2D MEF2D or DAZAP1/MEF2D allowed 3T3 cells to proliferate responsible for protein dimerization, DNA or RNA binding, under low serum (0.5%) conditions and suppressed apoptosis. transcriptional activation and subcellular localization, and might In contrast, MEF2D/DAZAP1 expression did not facilitate therefore contribute to leukemogenesis by altering signaling proliferation in low serum and led to a modest increase in pathways normally regulated by wild-type MEF2D and/or apoptosis. Both MEF2D/DAZAP1 and DAZAP1/MEF2D have DAZAP1. This study was undertaken to evaluate whether one oncogenic properties, and co-expression of both fusion proteins is synergistic. or both of MEF2D and DAZAP1 protein fusions have transform- Leukemia (2007) 21, 2470–2475; doi:10.1038/sj.leu.2404962; ing properties when ectopically expressed in NIH 3T3 cells. published online 27 September 2007 Keywords: leukemogenesis; chromosomal translocation; oncogenic transformation Materials and methods Cell culture cDNA fragments encoding human DAZAP1, MEF2D, DAZAP1/ MEF2D and MEF2D/DAZAP1 were FLAG-tagged5 and cloned Introduction into the XhoI site of pMSCVneo or pMSCVhyg vectors (Clontech, Mountain View, CA, USA). A transient–transfection 8 Chromosome translocations, which are often associated with system was used to create retroviruses harboring the DAZAP1 specific subtypes of leukemias, lymphomas and sarcomas, result and MEF2D wild-type and fusion cDNAs. Low-passage NIH 3T3 in aberrant expression of intact proteins or the generation of cells (ATCC) were infected with viral supernatant, expanded for À1 chimeric proteins that contribute to malignant transformation. 48 h then selected with 0.3–0.6 mg ml G418 sulfate (Invitro- À1 For most reciprocal translocations, only one protein from the gen Life Technologies, Carlsbad, CA, USA) and/or 0.2 mg ml two derivative chromosomes is expressed, or, if proteins from Hygromycin B (Invitrogen) for 2 to 3 weeks to achieve both derivatives are expressed, only one contributes signifi- stable transduction. To co-express both fusion proteins, retro- cantly to malignant transformation. However, in some hemato- viruses containing different antibiotic resistance markers were logical malignancies, reciprocal derivative fusion proteins are employed. expressed and both may play a role in transformation and/or the clinical phenotype of disease.1 One prominent example is Protein immunoprecipitation and western blot provided by PML-RARA and RARA-PML in acute promyelocytic Immunoprecipitation of FLAG-tagged proteins was performed leukemia.2,3 Transgenic mouse models of acute promyelocytic by consecutive use of the FLAG Tagged Protein IP kit (Sigma, St. leukemia suggest that expression of the reciprocal RARA-PML Louis, MO, USA) and the One-Step Complete IP-Western kit protein influences disease penetrance, phenotype and treatment (GenScript, Piscataway, NJ, USA). Following separation on response. sodium dodecyl sulfate-polyacrylamide gels, protein expression Recently, our laboratory and others reported that a variant was determined by immunoblot analysis using the M2 anti- t(1;19)(q23;p13.3) present in the TS-2 acute lymphoblastic FLAG primary antibody (Sigma) and goat-anti-mouse Ig-HRP leukemia cell line fuses the 19p13 gene DAZAP1 to the 1q23 secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, gene MEF2D leading to expression of reciprocal in-frame USA). The chemiluminescent signal was detected using the ECL Plus detection kit (Amersham, Arlington Heights, IL, USA). Correspondence: Dr V Prima, Department of Pediatric Hematology/ Oncology, University of Florida College of Medicine, PO Box 103633, Gainesville, FL 32610-3633, USA. E-mail: vprima@ufl.edu Soft agar assay Received 24 July 2007; revised 27 August 2007; accepted 28 August Stably transduced cells were trypsinized, suspended in Dulbec- 2007; published online 27 September 2007 co’s modified Eagle’s medium (Mediatech, Herndon, VA, USA) Cooperative transformation by MEF2D/DAZAP1 and DAZAP1/MEF2 V Prima and SP Hunger 2471 containing 0.3% agar and 15% calf serum (CS), plated onto a bottom layer containing 0.6% agar and covered with a top layer containing 0.6% agar. The cells were plated at a density of 3 Â 104 per well in a six-well plate, and fed with 1 ml of liquid media weekly. Colonies were stained with crystal violet and counted with ImageQuant TL software (Amersham Biosciences; Piscataway, NJ, USA) 14–17 days following plating, with software parameters adjusted to count colonies consisting of at least 100 cells. Adherent cell proliferation assay Cells in Dulbecco’s modified Eagle’s medium containing 0, 0.5 or 5% CS were dispensed into 24-well plates (5 Â 103 cells per well). Cell proliferation was assessed by WST-1-based colori- metric assay at 450 nm (Roche Diagnostics, Roswell, GA, USA). Apoptosis assay Cells in Dulbecco’s modified Eagle’s medium containing 5% CS were seeded at a concentration of 3 Â 104 cells per well and cultured overnight. Cells were incubated with 0.5% CS for 5 h, with 5% ethanol (positive control) for 1 h, or left untreated (negative control). Apoptosis was assessed with the APOPer- centage assay kit (Accurate Chemical, Westbury, NY, USA). Cells were stained with APOPercentage dye, lysed in 0.1N NaOH, and absorbance values were read at 550 nm. Results and discussion We established NIH 3T3 lines stably transduced with expression vectors encoding wild-type MEF2D and DAZAP1 proteins and the chimeric MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins (Figure 1a).5 FLAG-tagged recombinant proteins of the predicted size were expressed abundantly immediately follow- ing transient transfection of packaging Phoenix cells (Figure 1b). Figure 1 MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins result from shuffling MEF2D and DAZAP1 functional domains. (a) While all transduced 3T3 cells display neomycin or hygromycin Schematic of the wild-type and fusion proteins indicating the relative resistance following antibiotic selection, the levels of recombi- location of structural and functional domains. Arrows indicate fusion nant proteins expressed following 2 to 3 weeks of selection points of chimeric proteins. (b) Western blot of transiently transfected varied widely, presumably due to differential toxicities of the packaging cell lysates probed with anti-FLAG antibodies (DM, MD expressed proteins, with MEF2D/DAZAP1 showing the lowest and DM þ MDÀ cells transduced correspondingly with DAZAP1/ level of expression (Figure 1c). We were able to detect all the MEF2D, MEF2D/DAZAP1, or both fusion constructs). (c) Western blot of stably transduced 3T3 cell lysates probed with anti-FLAG antibodies recombinant proteins in 3T3 cell lysates using anti-FLAG (arrows indicate the ectopically expressed FLAG-tagged proteins). Act, immunoprecipitation (Figure 1d). actin. (d) Immunoprecipitation of FLAG-tagged proteins from 3T3 cell There were striking phenotypic consequences of ectopically lysates. expressed fusion proteins, which were most apparent when MEF2D/DAZAP1 and DAZAP1/MEF2D were co-expressed. Morphologically, NIH 3T3 cells expressing both DAZAP1/ DAZAP1/MEF2D or MEF2D/DAZAP1 led to approximately MEF2D and MEF2D/DAZAP1 fusion proteins display a dramatic 20-fold more colonies than background. The fact that MEF2D/ loss of the regular flat, surface-adherent fibroblast phenotype DAZAP1 transformed 3T3 cells despite very low protein observed in the parental cells (Figure 2a vs. b). As MEF2D is expression as assessed via western blot (Figure 1c) is reminiscent known to be a direct transcriptional activator of actin,9 it is of mixed lineage leukemia fusion proteins that immortalize possible that MEF2D fusion proteins alter actin microfilament stably transduced murine cells even though protein levels are polymerization, with resultant changes in cytoskeleton organi- undetectable by western blotting.12 Most notably, approxi- zation leading to changes in cell shape, motility and adhesion mately 60-fold more colonies than background
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