Leukemia (2010) 24, 1001–1011 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu ORIGINAL ARTICLE Effects of the NUP98–DDX10 oncogene on primary human CD34 þ cells: role of a conserved helicase motif ER Yassin, AM Abdul-Nabi, A Takeda and NR Yaseen Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA NUP98 gene rearrangements occur in acute myeloid leukemia In particular, the effects of NUP98–DDX10 have not been and result in the expression of fusion proteins. One of the most reported in either mice or in human primary cells. DDX10 is a frequent is NUP98–DDX10 that fuses a portion of NUP98 to a putative RNA helicase of unknown function that contains eight portion of DDX10, a putative DEAD-box RNA helicase. Here, we 7,8 show that NUP98–DDX10 dramatically increases proliferation highly conserved motifs typical of DEAD box RNA helicases. 8 and self-renewal of primary human CD34 þ cells, and disrupts It is thought to be involved in ribosome assembly. NUP98– their erythroid and myeloid differentiation. It localizes to DDX10 consists of the entire FG repeat region of NUP98 fused their nuclei and extensively deregulates gene expression. to a C-terminal portion of DDX10 containing two of the eight Comparison to another leukemogenic NUP98 fusion, NUP98– conserved helicase motifs. HOXA9, reveals a number of genes deregulated by both oncoproteins, including HOX genes, COX-2, MYCN, ANGPT1, Here, we report on the in vitro transforming effects of REN, HEY1, SOX4 and others. These genes may account for the NUP98–DDX10 in primary human CD34 þ hematopoietic similar leukemogenic properties of NUP98 fusion oncogenes. stem/progenitor cells and the underlying changes in global The YIHRAGRTAR sequence in the DDX10 portion of NUP98– gene expression, and provide evidence that a conserved DDX10 represents a major motif shared by DEAD-box RNA helicase motif has a role in these effects. This is the first helicases that is required for ATP binding, RNA-binding and demonstration of the effects of NUP98–DDX10 and the helicase functions. Mutating this motif diminished the in vitro transforming ability of NUP98–DDX10, indicating that it has a first report of the effects of a non-homeodomain NUP98 fusion role in leukemogenesis. These data show for the first time the in primary human cells. in vitro transforming ability of NUP98–DDX10 and show that it is partially dependent on one of the consensus helicase motifs of DDX10. They also point to common pathways that may Materials and methods underlie leukemogenesis by different NUP98 fusions. Leukemia (2010) 24, 1001–1011; doi:10.1038/leu.2010.42; Plasmid construction published online 25 March 2010 The HA-tagged NUP98–DDX10 cDNA was generated by Keywords: DDX10; NUP98–DDX10; helicase; AML PCR and subcloned into EcoRI/XbaI sites of pTracer-CMV/Bsd. The NUP98–DDX10/3Q mutant was created by replacing an EagI/HindIIII fragment with a synthetic fragment containing three arginine to glutamine mutations in the YIHRAGRTAR motif. HA-tagged NUP98–DDX10 and NUP98–DDX10/3Q Introduction were subcloned upstream of IRES into the HpaI site of MSCV– IRES–GFP. PCR products and mutations were confirmed by At least 21 rearrangements of the NUP98 gene have been sequencing. The KBTBD10 and PLN luciferase constructs were described in myeloid malignancies including acute myeloid previously described.9 leukemia (AML), myelodysplastic syndrome (MDS) and blast crisis of chronic myelogenous leukemia.1,2 These rearrange- ments result in fusion of an N-terminal segment of NUP98 to a Retrovirus production C-terminal segment of a fusion partner. The inv(11)(p15q22) GP293 cells were transiently transfected with 4.4 mg of retroviral rearrangement that has been reported in AML, MDS and chronic vector and 1.1 mg of pVSV-G expression vector using myelogenous leukemia blast crisis results in the expression Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA, USA). of NUP98–DDX10, one of the most common NUP98 fusions. After 48 h, the culture supernatant, containing VSV-G-pseudo- It shows a particularly strong association with therapy-related typed retrovirus, was collected and used for transduction of PG13 packaging cells by spinoculation in the presence of AML and MDS, especially in patients who had been treated with –1 topoisomerase II inhibitors.1 8 mgml polybrene (Hexadimethrine Bromide; Sigma-Aldrich About half of the reported NUP98 fusion partners are Corp., St Louis, MO, USA). The PG13 culture supernatant homeodomain-containing transcription factors. The resulting containing GaLV-pseudotyped retrovirus was used for transduc- fusions are believed to act as aberrant transcription factors.3–6 tion of CD34 þ primary cells. The remaining NUP98 fusion partners are a heterogeneous group and their role in leukemogenesis is not well understood. Immunofluorescence microscopy Cells were centrifuged onto a slide using a Cytospin centrifuge Correspondence: Dr NR Yaseen, Department of Pathology and (Thermo Fisher Scientific, Waltham, MA, USA), fixed with 4% Immunology, Campus Box 8118, Washington University School of paraformaldehyde in D-PBS for 20 min and permeabilized with Medicine, St Louis, MO 63110, USA. E-mail: [email protected] 0.1% Triton X-100 for 20 min at room temperature. Two percent Received 9 September 2009; revised 3 December 2009; accepted 22 of normal donkey serum in D-PBS with 0.1% Tween 20 was January 2010; published online 25 March 2010 used for blocking and washing. Anti-HA antibody (12CA5; Effects of NUP98–DDX10 on human CD34 þ cells ER Yassin et al 1002 Roche Applied Science, Indianapolis, IN, USA) and Alexa Fluor Table 1 Genes deregulated by both NUP98-DDX10 and NUP98- 647-conjugated anti-mouse IgG (Invitrogen) were used for HOXA9 with possible roles in cell transformation staining. DAPI (40,6-diamidino-2-phenylindole, dihydrochloride; Invitrogen) at 300 nM was used for nuclear counterstain. Images Fold change (NUP98-DDX10) were captured with an Eclipse 80i fluorescent microscope 6 h 3 days 8 days (Nikon, Melville, NY, USA) using MetaMorph 6.3r2 software (MDS Analytical Technologies, Downington, PA, USA) and Upregulated genes pseudo-colored. Brightness and contrast were adjusted and HOXA3 3.6 9.6 images were superimposed using Adobe Photoshop CS4, HOXA5 3.7 10.2 version 11.0 for Mac OS (Adobe Systems, San Jose, CA, USA). HOXA6 3.7 4.1 HOXA7 2.9 3.9 HOXA9 2.1 2.5 2.3 HOXB3 3.8 14.6 Retroviral transduction and analysis of primary HOXB5 6.1 3.0 human CD34 þ cells HOXB6 5.9 13.6 Frozen human CD34 þ cells purified from mobilized peripheral HOXB7 3.6 4.5 blood of two healthy volunteers were purchased from the MEIS1 2.6 14.9 Fred Hutchinson Cancer Research Center (Seattle, WA, USA). PBX3 1.8 EGR1 3.1 3.0 Cells were prestimulated and transduced with retrovirus as 10 SOX4 2.1 described. After 46 h, GFP-positive cells were isolated using MYCN 3.1 a FACSVantage s.e. (BD Biosciences, San Jose, CA, USA) and ZNF521 2.3 5.5 expression of the transfected gene was confirmed by immuno- HEY1 2.7 blotting with anti-HA antibody. Long-term liquid culture of PTGS2 3.0 primary cells was performed in the presence of a cytokine REN 94.8 50.6 10 ANGPT1 3.9 cocktail as previously described. Colony-forming cell (CFC) RYK 2.1 and long-term culture-initiating cell (LTC-IC) assays were STYK1 2.4 performed as previously described.10 Cytospin preparations of cells harvested from the CFC plates were stained with Giemsa Downregulated genes and a 500-cell differential count was performed using an RAP1A À21.9 À20.7 Olympus BX51 microscope (Olympus America, Center Valley, FCGR2C 12.5 CLEC7A À3.1 PA, USA). Photomicrographs were taken with an Olympus ALOX5 À2.2 DP71 camera with a  60 oil objective. FPR3 À3.0 A2M À2.6 ELA2 À2.2 Flow cytometry GADD45B À2.2 Flow cytometry was performed on a FACScan flow cytometer MS4A3 À2.6 TRIM35 À2.4 upgraded to five colors and two lasers (BD Biosciences) and a ZFHX3 À7.6 FACSVantage s.e. flow sorter with three lasers and eight NDRG2 À1.9 detectors (BD Biosciences), and analyzed using FlowJO v7.2.4 RAD18 À3.7 software (Tree Star Inc., Ashland, OR, USA). The antibodies EED À2.3 used for these studies were CD11b (phycoerythrin-conjugated clone ICRF44) from eBioscience (San Diego, CA, USA); CD235a (allophycocyanin-conjugated clone GA-R2) from BD (Franklin only). Probes scored as increasing and absent in the numerator, Lakes, NJ, USA); and CD33 (allophycocyanin-conjugated and those scored as decreasing and absent in the denominator clone D3HL60.251) and CD45 (phycoerythrin-Cy7-conjugated were also filtered out. Probe sets induced or repressed by clone J.33) from Beckman Coulter (Miami, FL, USA). 1.74 fold or greater in two independent experiments were considered differentially expressed. Fold changes shown in Table 1 represent the average of all probesets recognizing Microarray analysis of primary human CD34 þ cells each gene in both experiments. For the 6-h time point, cells were subjected to nucleofection with either control pTracer-CMV/Bsd plasmid or with plasmid- expressing NUP98–DDX10 or NUP98–HOXA9 as previously Luciferase assay described.10 For the 3-day and 8-day time points, cells Cells (107) were transfected by electroporation using a Bio-Rad were retrovirally transduced as described above. GFP-sorted GenePulser (Bio-Rad, Hercules, CA, USA) with 10 mg pGL4.11 cells were snap-frozen and submitted to the Siteman Cancer vector or pGL4.11 driven by the indicated promoters and 20 mg Center Laboratory for Clinical Genomics where total RNA was of either empty pTracer-CMV/Bsd vector, vector expressing isolated and target preparation and microarray hybridization HA-tagged NUP98–DDX10 or HA-tagged NUP98–DDX/3Q.