Mutations in the nucleolar phosphoprotein, nucleophosmin, promote the expression of the oncogenic transcription factor MEF/ELF4 in leukemia cells and potentiates transformation

Abstract

Myeloid ELF1-like factor (MEF/ELF4), a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers. Here, we report a mechanism by which MEF/ELF4 may be activated by a common leukemia-associated mutation in the nucleophosmin gene. By using a tandem affinity purification assay, we found that MEF/ELF4 interacts with multifactorial protein nucleophosmin (NPM1). Coimmunoprecipitation and GST pull-down experiments demonstrated that MEF/ELF4 directly forms a complex with NPM1 and also identified the region of NPM1 that is responsible for this interaction. Functional analyses showed that wild-type NPM1 inhibited the DNA binding and transcriptional activity of MEF/ELF4 on the HDM2 promoter, whereas NPM1 mutant protein (Mt-NPM1) enhanced these activities of MEF/ELF4. Induction of Mt-NPM1 into MEF/ELF4-overexpressing NIH3T3 cells facilitated malignant transformation. In addition, clinical leukemia samples with NPM1 mutations had higher human MDM2 (HDM2) mRNA expression. Our data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in MEF/ELF4-dependent leukemogenesis.

FIGURE 1.

NPM1 interacts with MEF/ELF4. A, 293T cells were transfected with the indicated expression plasmids. After 48 h, cell lysates were immunoprecipitated (IP) with anti-FLAG and anti-V5 antibodies. Immunoprecipitates were analyzed by 10% SDS-PAGE and subjected to immunoblotting (WB) with anti-V5 antibody (top row) or anti-FLAG antibody (bottom row). B, MEF/ELF4 interacts directly with NPM1 in vitro. In vitro association assays were undertaken by incubating His-MEF/ELF4 fusion protein immobilized by using a His-column with biotin-labeled MEF/ELF4 (lane 1). His alone was incubated with biotin-labeled NPM1 (lane 2) as a control. C, NPM1 structure and the relative binding of MEF/ELF4 (schematic). HomoD, homodimerization domain, residues 1–117; AD/NLS, acidic domain/nuclear localization sequence, residues 117–187; HeteroD, heterodimerization domain, residues 187–259; NBD, nucleic acid binding domain, residues 259–294. D, the N-terminal portion of NPM1 is the MEF/ELF4-interacting domain. Bacterially expressed and purified GST, GST-NPM1, and GST-NPM1 mutants with deletions were mixed with bacterially expressed and purified His or His-MEF/ELF4 protein. Recombinant proteins were subjected to His or GST affinity columns, followed by immunoblotting with anti-GST or anti-His antibodies. a, the reactive samples were subjected to analyses on a His affinity column followed by immunoblotting with anti-His antibodies (bottom left) or with anti-GST antibodies (top left). b, the reactive samples were subjected to GST affinity columns, followed by immunoblotting with anti-GST antibodies (top right) or with anti-His antibodies (bottom right).

FIGURE 2.

EMSA with recombinant His-MEF/ELF4, His, GST, and GST-Wt-NPM1. His-MEF/ELF4 was incubated with GST and GST-Wt-NPM1 at room temperature prior to EMSA by using a biotin-conjugated APET probe (lanes 1–4). An excess amount of unlabeled APET competitor was added to the reaction mixtures (lanes 5 and 6).

FIGURE 3.

Wt-NPM1 inhibits, whereas Mt-NPM1 enhances, MEF/ELF4-dependent APET promoter transactivation. 293T human kidney (A), COS7 monkey kidney (B), and U937 human hematological (C) cell lines were co-transfected with the luciferase reporter gene of an artificial MEF/ELF4 target promoter (APET) and effector genes. The target promoter and effector genes were as follows: pGL4/APET (lane 1); pGL4/ETSm-APET (lane 2); pGL4/APET and pcDNA/MEF/ELF4 (lane 3); pGL4/APET, pcDNA/MEF/ELF4, and pcDNA/Wt-NPM1 (lane 4); pGL4/ETSm-APET and pcDNA/MEF/ELF4 (lane 5); and pGL4/APET and pcDNA/Wt-NPM1 (lane 6). Luciferase activity by pGL4/APET alone was assigned a value of 1.0. The analysis was performed in triplicate assays, and the results were reproducible. The results are shown as the mean ± S.D. (error bars). *, p < 0.05. D, 293T cells transduced with siRNA encoding vector (siWt-NPM1) were harvested 72 h after transduction for Western blotting. Hsp90 is shown as a control. sicNPM1, control siRNA non-relevant to the expression of NPM1; Wild, without transduction. E, 293T cells were co-transfected with the luciferase reporter plasmid (pGL4/APET), expression plasmid (pcDNA MEF/ELF4), and siWt-NPM1 gene (pcDNA/siRNA-Wt-NPM1) or control. Luciferase activity by pGL4/APET alone was assigned a value of 1.0. The analysis was performed in triplicate assays, and the results were reproducible. The results are shown as the mean ± S.D. *, p < 0.05. F, 293T cells were co-transfected with the luciferase reporter gene of an artificial MEF/ELF4 target promoter and effector genes. Target promoter and effector genes were as follows: pGL4/APET (lane 1); pGL4/APET and pcDNA/MEF/ELF4 (lane 2); pGL4/APET, pcDNA/MEF/ELF4, and Wt-NPM1 (lane 3); pGL4/APET, pcDNA/MEF/ELF4, and Mt-A-NPM1, Mt-I-NPM1, or Mt-J-NPM1 (lanes 4–6, respectively); and pGL4/APET and pcDNA/Wt-NPM1, Mt-A-NPM1, Mt-I-NPM1, or Mt-J-NPM1 (lanes 7–10, respectively). Luciferase activity by pGL4/APET alone was assigned a value of 1.0. The analysis was performed in triplicate assays, and the results were reproducible. The results are shown as the mean ± S.D. *, p < 0.05. G, COS7 cells were co-transfected with the luciferase reporter gene of an artificial MEF/ELF4 target promoter and effector genes. The target promoter and effector genes were as follows: pGL4/APET (lane 1); pGL4/APET and pcDNA/MEF/ELF4 (lane 2); pGL4/APET, pcDNA/MEF/ELF4, and Wt-NPM1 (lane 3); pGL4/APET, pcDNA/MEF/ELF4, and Mt-A-NPM1 or Mt-I-NPM1 (lanes 4 and 5, respectively); and pGL4/APET and pcDNA/Wt-NPM1, Mt-A-NPM1, or Mt-I-NPM1 (lanes 6–8, respectively). Luciferase activity by pcDNA/APET alone was assigned a value of 1.0. The analysis was performed in triplicate assays, and the results were reproducible. The results are shown as the mean ± S.D. (*, p < 0.05). H, U937 cells were co-transfected with the luciferase reporter gene of an artificial MEF/ELF4 target promoter and effector genes. Target promoter and effector genes were as follows: pGL4/APET (lane 1); pGL4/APET and pcDNA/MEF/ELF4 (lane 2); pGL4/APET, pcDNA/MEF/ELF4, and Wt-NPM1 (lane 3); pGL4/APET, pcDNA/MEF/ELF4, and Mt-A-NPM1 (lane 4); and pGL4/APET and pcDNA/Wt-NPM1 or Mt-A-NPM1 (lanes 5 and 6, respectively). Luciferase activity by pcDNA/APET alone was assigned a value of 1.0. The analysis was performed in triplicate assays, and the results were reproducible. The results are shown as the mean ± S.D. *, p < 0.05. I, 293T cells were co-transfected with 0.1 μg of the luciferase reporter gene of an artificial MEF/ELF4 target promoter (lanes 1–9) and 0.1 μg of effector genes (pcDNA/MEF/ELF4) (lanes 1–6). The effector genes were as follows: 0.2 μg of Mt-A-NPM1 (lane 1); 0.16 μg of Mt-A-NPM1 and 0.04 μg of Wt-NPM1 (lane 2); 0.1 μg of Mt-A-NPM1 and 0.1 μg of Wt-NPM1 (lane 3); 0.04 μg of Mt-A-NPM1 and 0.16 μg of Wt-NPM1 or 0.2 μg of Wt-NPM1 (lanes 4 and 5, respectively); none (lane 6); pGL4/APET and 0.2 μg of Mt-A-NPM1 (lane 7); pGL4/APET and 0.2 μg of Wt-NPM1 (lane 8); and pGL4/APET (lane 9). Luciferase activity by pGL4/APET alone was assigned a value of 1.0. The analysis was performed in triplicate assays, and the results were reproducible. The results are shown as the mean ± S.D. *, p < 0.05.

FIGURE 4.

Mt-A-NPM1 does not interact with MEF/ELF4 in vivo. 293T cells were transfected with the indicated expression plasmids. After 48 h, cell lysates were immunoprecipitated (IP) with anti-FLAG and anti-V5 antibodies. Immunoprecipitates were analyzed by 10% SDS-PAGE and subjected to immunoblotting (WB) with anti-V5 antibody (top row) or anti-FLAG antibody (bottom row).

FIGURE 5.

Localization of MEF/ELF4 was unaffected by the mutation of NPM1. A, 293T cells were transfected with the GFP-MEF/ELF4 fusion protein expression vector and pcDNA/V-Wt-NPM1 (a) or pcDNA/V-Mt-A-NPM1 (b). Forty-eight hours after transfection, cells were fixed and immunofluorescence-stained with anti-V tag antibody. B, Western blotting of FLAG-MEF/ELF4 subcellular distribution in 293T cells co-transfected with pFLAG-MEF/ELF4 and pcDNA/V-Wt-NPM1 or pcDNA/V-Mt-A-NPM1. Purity of the subcellular fractions was assessed by blotting with histone H1 (nuclear extraction) and Hsp70 (cytoplasmic extraction).

FIGURE 6.

Mt-NPM1 stimulates MEF/ELF4-induced hyperproliferation and transformation. NIH3T3 cells transfected with various combinations of expression plasmids were plated in soft agar on 60-mm dishes and incubated for 2 weeks. A, microscopy of MEF/ELF4-transfected NIH3T3 cells with Wt-NPM1 or Mt-A-NPM1. B, the average number of colonies of three independent experiments with S.D. (error bars). *, p < 0.05.

FIGURE 7.

MEF/ELF4 transactivates the HDM2 promoter. A, MEF/ELF4 binds to the HDM2 promoter in vivo. FLAG-MEF/ELF4-bound DNA from 293T cells was immunoprecipitated with FLAG antibody or normal mouse IgG. RQ-PCR amplification was performed on the corresponding templates by using primers for HDM2. B, structure of the HDM2 promoter region (−82 to −122) (schematic). C, 293T cells were transfected with HDM2 promoter-driven luciferase reporter plasmid encoding wild-type (B (a)) or mutant (B (b)) protein. Luciferase activity by pcDNA alone was assigned a value of 1.0. The analysis was performed in triplicate assays, and the results were reproducible. The results are shown as the mean ± S.D. (error bars). D, 293T cells were co-transfected with pFLAG/MEF/ELF4 and pcDNA/Wt-NPM1 or pcDNA/Mt-A-NPM1. RQ-PCR amplification was undertaken on corresponding templates using primers for HDM2. The analysis was performed in triplicate assays, and the results were reproducible. The results are shown as the mean ± S.D. *, p < 0.05.

FIGURE 8.

Expression of Mt-NPM1 and higher expression of MEF/ELF4 are associated with the elevated expression of HDM2 in CD34-positive AML cells. Total RNA isolated from 22 AML patients (CD34-positive leukemia cells) was analyzed for the expression of HDM2 by RQ-PCR. Shown is stratification by the presence of the NPM1 mutation (A) and by the level of ELF4/MEF (B). These bars were median lines for each group. *, p < 0.009 against Wt-NPM1; **, p < 0.03 against MEF/ELF4-L, assessed by analysis of variance followed by Scheffe's multiple comparison test.