Original Paper | Published:

The serine-threonine kinase MNK1 is post-translationally stabilized by PML-RARα and regulates differentiation of hematopoietic cells

Abstract

Microarray analyses were performed to identify target genes that are shared by the acute myeloid leukemia (AML) translocation products PML-RARα, PLZF-RARα and AML1-ETO in inducibly transfected U937 cell lines. The cytoplasmic serine and threonine kinase MNK1 was identified as one of the target genes. At the protein level, MNK1 was significantly induced by each of the three fusion proteins. Protein half-life analyses showed that PML-RARα enhanced MNK1 protein stability in U937 cells and ATRA exposure decreased MNK1 half-life in NB4 cells. EIF4E, the main MNK1 substrate, plays a role in the pathogenesis of a variety of cancers. Upon MNK1 overexpression, eIF4E phosphorylation increased as a sign of functional activation. Interestingly, MNK1 protein expression decreased during myeloid differentiation. Inhibition of MNK1 activity by a specific inhibitor (CGP57380) enhanced differentiation of HL60 and 32D cells, further suggesting a role for MNK1 in the myeloid differentiation. In addition, kinase dead mutants of MNK1 significantly impaired proliferation of 32D cells. Immunohistochemistry of primary AML bone marrow biopsies showed strong cytoplasmic MNK1 expression in 25 of 99 AML specimens (25%). MNK1 expression was associated with high levels of c-myc expression. Taken together, we identified MNK1 as a target gene of several leukemogenic fusion proteins in AML. MNK1 plays a role in myeloid differentiation. These data suggest a role for MNK1 in the AML fusion protein-associated differentiation block.

Introduction

In acute myeloid leukemia (AML), balanced translocations encode aberrant transcription factors, which influence myeloid differentiation and proliferation. Acute promyelocytic leukemia (APL) is associated with the t(15;17) and rarely the t(11;17) chromosomal translocations resulting in the fusion proteins PML-RARα and PLZF-RARα (Kalantry et al., 1997). These fusion proteins disrupt the normal functions and recruit histone deacetylases (HDACs) to promoters of target genes (Grignani et al., 1998). The aberrant recruitment of HDACs to the promoter of target genes is also a feature of the AML1-ETO fusion protein resulting from t(8;21) (Amann et al., 2001; Yuan et al., 2001). Thus, the proteins encoded by the genes involved in these translocations are thought to function as direct or indirect regulators of transcription. The functional similarities of these translocation products in inducing a block in hematopoietic differentiation suggest similar pathogenetic mechanisms.

On the basis of this knowledge, we used microanalyses to identify shared target genes of PML-RARα, PLZF-RARα and AML1-ETO in prior work (Muller-Tidow et al., 2004b). We could identify 63 genes that were significantly regulated, with 52 being repressed and 11 being induced by all three AML fusion proteins. Most of these genes could be verified by real time RT–PCR to be regulated at the mRNA level (Muller-Tidow et al., 2004b).

The mitogen-activated protein kinase MNK1 (MAP kinase-interacting kinase1) was identified as one of the fusion protein target genes.

MNK1, a serine/threonine kinase, is phosphorylated and thereby activated through both the extracellular signal-regulated protein kinases (ERKs) and p38 MAP kinase. MNK1 integrates signals emanating from both MAP kinase pathways to phosphorylate relevant substrates (Fukunaga and Hunter, 1997; Morley and McKendrick, 1997; Waskiewicz et al., 1997; Wang et al., 1998). Treatment with peptide growth factors, phorbol esters or UV irradiation induce post-translational modification and enzymatical activation of MNK1 (Waskiewicz et al., 1997). Stimulated MNK1 phosphorylates the cap-binding eucaryotic initiation factor 4E (eIF4E) at the physiologically relevant site, Ser 209, in response to mitogens and cellular stress (Waskiewicz et al., 1999). This phosphorylation activates the eIF4E protein in cells.

The eIF4E is the rate-limiting component of cap-dependent translation initiation. It binds the methyl-7-guanosine cap at the 5′ UTR of processed mRNA and transports the transcript to the ribosome. Its tightly regulated activity in cells is critically important in cell growth and transformation as eIF4E promotes inappropriate translation of mRNA (Lazaris-Karatzas et al., 1990; Sonenberg and Gingras, 1998; Topisirovic et al., 2003b).

Additionally, eIF4E has been implicated in the pathogenesis of various types of cancer (Sorrells et al., 1999; Nathan et al., 2000; Dua et al., 2001). Even moderate overexpression of eIF4E causes dysregulated proliferation and tumorigenic transformation in immortalized cell lines (Lazaris-Karatzas et al., 1990; Lazaris-Karatzas et al., 1992; Lazaris-Karatzas and Sonenberg, 1992).

Here, we demonstrate that MNK1 protein is induced by PML-RARα, PLZF-RARα and AML1-ETO. MNK1 is highly expressed in about 25% of all AMLs and functions in myeloid differentiation. Our data show evidence for a role of MNK1 in the fusion protein-induced differentiation block.

Results

Decrease of MNK1 mRNA by AML fusion proteins

MNK1, a serine/threonine protein kinase, was identified in microarray analyses to be expressed at a 2.5–3-fold lower level in PML-RARα, PLZF-RARα and AML1-ETO-expressing U937 cells (Figure 1a, (Muller-Tidow et al., 2004b)). In order to confirm these results, the same cell lines were incubated with 0.1 mM zinc to induce fusion proteins as described (Muller-Tidow et al., 2004b). RNA and cDNA were prepared and real-time RT–PCR experiments performed. MNK1 mRNA levels were significantly lower in PML-RARα and PLZF-RARα-positive U937 cells compared to U937-control cells that contain the empty vector PMT (Figure 1b). In U937-AML1-ETO cells, MNK1 mRNA levels did not differ from the expression levels detected in U937-control cells.

Figure 1
figure1

Regulation of MNK1 mRNA levels by AML fusion proteins. (a) Total mRNA was prepared from zinc-induced U937 cell lines expressing different fusion proteins or empty vector. The microarray analyses identified MNK1 as one of the genes repressed at the mRNA level by the AML fusion proteins. Bars indicate the average intensity units (Affymetrix Microarray Suite software) following data normalization to 2500 average intensity units. (b) For confirmation purposes, MNK1 mRNA levels were analysed by real-time quantitative RT–PCR using TaqMan technology in U937 cell lines induced to express the indicated fusion proteins. Means and standard error of five independent experiments are indicated

Induction of MNK1 protein by AML fusion proteins and its expression in leukemia cell lines

Next, we tested the influence of all three fusion proteins on the protein level of MNK1. Therefore, we analysed MNK1 expression at the protein level by Western blot after induction of the fusion proteins (Figure 2a). Surprisingly, in contrast to the findings at the mRNA level, MNK1 was induced at the protein level by all three fusion proteins (also in zinc-induced U937-AML1-ETO cells, data not shown). In U937 cells, expression of the leukemogenic fusion proteins was associated with an increase in MNK1 protein expression (Figure 2a). Since this finding was unexpected, the experiment was performed more than five times with different lysates and different cells. In all these experiments, PML-RARα and PLZF-RARα-expressing cells consistently induced MNK1 protein expression. In order to verify that MNK1 protein expression is due to the fusion proteins and not stress induced, we performed cell cycle analyses as shown in Figure 2e. The results verify that at least in the case of PML-RARα, MNK1 induction is a consequence of fusion protein expression as no significant changes in cell cycle could be observed compared to the control cells containing the empty vector. Also, Western blot analyses in Figure 3d show that rapamycin, a reagent that impairs cell cycle progression, did not alter MNK1 stability.

Figure 2
figure2

MNK1 protein is induced by AML fusion proteins and induces phosphorylation of eIF4E in myeloid cells. (a) Zinc-induced AML fusion protein-expressing cell lines or control U937 cells were analysed for MNK1 protein expression by Western blot analyses. Actin served as loading control. (b) Tetracycline inducible AML1-ETO U937 cells also expressed high levels of MNK1, whereas normal U937 cells did not. MNK1 was also expressed in several myeloid leukemic cell lines. (c) HL60 cells were exposed to TPA and cells were harvested at the indicated time points. Protein lysates were prepared and analysed for MNK1 expression. Nonexposed cells served as control. (d) MNK1-expression vector or pCDNA3.1 vector were transfected into 32D cells. Overexpression of MNK1 was verified by Western blotting. Phosphorylated eIF4E was only detected in MNK1-overexpressing cells. Exposure of the cells to the MNK1 inhibitor CGP57380 (5 μ M) resulted in a strong decrease of phosphorylated eIF4E (peIF4E). (e) MNK1 expression did not occur due to general cell cycle arrest. Control U937 cells, U937-PML-RARα or U937-AML1-ETO cells were zinc-induced for 24 h. Cells were stained with Propidiumiodide and cell cycle profiles were analysed by flow cytometry

Figure 3
figure3

MNK1 protein half-life regulation by PML-RARα and ATRA. (a) U937 PMT control cells or U937-PML-RARα cells were zinc-induced for 24 h before cycloheximide was added in order to block protein synthesis. Cells were harvested at the indicated time points and analysed for MNK1 protein levels to determine the protein half-life. The bar diagram on the right indicates the average MNK1 protein level of the two independent experiments. For densitometry analyses, we used an INTAS camera (Epichem3 Darkroom) and the programme GelPro Analyser (1D-Gel ToolBar). (b) In a dose–response study, NB4 cells were exposed to different concentrations of ATRA. Cells were harvested after 24 h and Western blot analysis for MNK1 protein was performed. (c) To analyse the effects of ATRA on MNK1 protein half-life, NB4 cells were exposed to ATRA (10−6M) for 24 h. Then, cycloheximide was added and MNK1 protein levels were monitored by Western blotting. ATRA-exposed cells no longer had detectable MNK1 protein after a 12 h exposure to cycloheximide, but nonexposed NB4 cells still had detectable MNK1 levels after 24 h. (d) Cell cycle inhibition does not influence MNK1 expression in myeloid cells. HL60 cells were exposed to rapamycin at the indicated concentrations for 24 h. Western blots were performed to determine the effects of rapamycin on MNK1 protein levels

Since the Zn2+-inducible system seemed to be leaky and the induction of AML1-ETO altered the cell cycle distribution, we tested MNK1 expression in tet-inducible U937-AML1-ETO cells. Again Western blot analysis revealed increased MNK1 protein levels (Figure 2b). Significant levels of MNK1 protein were also found in several leukemic cell lines such as HL60, KCL-22 cells and NB4 cells, the latter naturally expressing PML-RARα. MNK1 protein was expressed at very low or nondetectable levels in nontransfected U937 and ML-1 cells (Figure 2b). These findings established widespread MNK1 protein expression in several leukemic cell lines and MNK1 induction by several fusion proteins.

We further analysed whether MNK1 was regulated during myeloid differentiation. HL60 cells, which expressed high levels of MNK1, underwent macrophage-like differentiation upon exposure to TPA (12-O-tetradecanoylphorbol 13-acetate) as confirmed by flow cytometry analysis (data not shown). MNK1 protein levels significantly decreased in TPA-exposed cells but not in control HL60 cells (Figure 2c). These experiments indicated that MNK1 was downregulated during myeloid TPA-induced differentiation.

Analysis of the association between elevated MNK1 protein levels and eIF4E phosphorylation

MNK1 is a protein involved in the regulation of protein translation and phosphorylates the eIF4E in vivo and in vitro (Waskiewicz et al., 1997; Pyronnet et al., 1999; Waskiewicz et al., 1999).

The eIF4E subunit of the eucaryotic initiation factor 4F (eIF4F) complex is present in limiting amounts (Hiremath et al., 1985; Duncan et al., 1987; Rau et al., 1996) and hence, the protein is the main regulatory factor of translation initiation. The eIF4E protein has been implicated in the pathogenesis of a broad spectrum of cancers (Sorrells et al., 1999; Nathan et al., 2000; Dua et al., 2001). For this reason, we analysed eIF4E expression and phosphorylation in 32D cells stably transfected with MNK1. Figure 2d depicts a Western blot of MNK1 overexpression in the MNK1 stable cell line compared to cells containing pcDNA3 as the empty control vector. To gain insight into a potential role of MNK1 in eIF4E phosphorylation, we employed a potent small molecular weight inhibitor (CGP57380), which was a kind gift of Dr Gram (Novartis, Knauf et al., 2001). Experiments performed in the presence and absence of the MNK1 inhibitor CGP57380 showed that the eIF4E translation factor was consistently detectable and its expression levels were not altered by MNK1 overexpression. However, phosphorylation of eIF4E increased in MNK1-overexpressing cells, demonstrating a direct link between MNK1 and eIF4E phosphorylation. Exposure of MNK1-transfected cells to 5 μ M CGP57380 decreased eIF4E phosphorylation, while overall eIF4E protein levels remained constant (Figure 2d). These data provide evidence that MNK1 can phosphorylate eIF4E in myeloid cells and activates its signaling cascade. The effects of CGP57380 make it very likely that the observed phosphorylation events are dependent on MNK1 kinase activity.

AML fusion proteins stabilize MNK1 protein and prolong its half-life

The conflicting MNK1 protein and mRNA expression data suggested that MNK1 regulation by AML fusion proteins occurred at the post-transcriptional level. We analysed whether AML fusion proteins exercised their effects by prolonging MNK1 half-life. Cycloheximide was added to either U937 or U937-PML-RARα cells after 24 h of zinc induction to block protein translation. Samples were sequentially harvested and analysed for MNK1 protein concentration by Western blot analyses (Figure 3a). MNK1 half-life was determined by calculating the intensity of the bands using densitometry (Figure 3a). Figure 3a depicts a representative experiment. MNK1 protein degradation did not follow first-order kinetics. Protein levels decreased slowly in the first hours but accelerated after 6–8 h. Importantly, PML-RARα prolonged the half-life especially in the later phases. At time 0, the relative amount of MNK1 in PML-RARα-positive U937 cells was 17 times higher than in U937-control cells. Following cycloheximide exposure, MNK1 levels were 84.3 and 51.8 times higher after 6 and 8 h in PML-RARα-positive cells (mean of two independent experiments). The overall half-life was about 50% longer.

Further experiments with NB4 cells indicated that all-trans retinoic acid (ATRA) decreased MNK1 protein levels in a dose-dependent manner (Figure 3b). In accordance with these findings, ATRA (10−6M) exposure shortened the protein half-life of MNK1 (Figure 3c, different experiment and exposure time than shown in Figure 3b).

32D cell proliferation depends on MNK1 function

Our data established MNK1 as a shared target gene of several AML fusion proteins. This finding along with MNK1 regulating an oncogenic translation factor suggested a potential role for MNK1 in hematopoiesis and leukemogenesis. We further analysed the functional role of MNK1 in hematopoietic cells using MNK1 and MNK1-mutant expression vectors. The 32D hematopoietic progenitor cells, which express low levels of endogenous MNK1 (Figure 2d), were transfected with either an inactive mutant MNK1 or MNK1-WT expression vector. Empty vector (pcDNA3)-transfected 32D cells served as control. MNK1-MA is a kinase-deficient mutant and MNK1-AA contains a mutation at a phosphorylation site (Knauf et al., 2001). Both MNK1 mutants are inactive in MNK1 function and could potentially function as dominant negative proteins. Following transfection with the MNK1 expression vectors, which harbor a neomycin-resistance gene, cells were seeded in colony assays. Wild-type MNK1 did not alter 32D cell clonal growth (Figure 4). In contrast, the MNK1 mutants (MNK1-MA and MNK1-AA) impaired colony formation of 32D cells by about 50% (Figure 4), whereas we could see nonsignificant effects on colony size. These data provided the first hint that MNK1 was important for hematopoietic cell growth.

Figure 4
figure4

MNK1 mutants block myeloid cell growth. Effects of pcDNA3.1 control, MNK1-WT or kinase inactive MNK1-mutants on colony-forming abilities were analysed. Electroporated 32D cells were plated in colony assays in the presence of selection antibiotics. The numbers of colonies formed by the kinase-deficient mutant MNK1 (MA) and the phosphorylation site mutant (AA) MNK1 were lower than the number of colonies formed by either control or MNK1-WT-transfected cells. Indicated are means and standard error of five independent experiments. The differences between the MNK1-MA mutant (P=0.043) but not of the MNK1-AA (P=0.08) reached statistical significance compared to the pCDNA3.1-transfected cells

Inhibition of MNK1 enhances differentiation

In hematopoietic cells, cell cycle control, proliferation and differentiation are closely linked. To gain insight into a potential role of MNK1 in myeloid differentiation, 32D cells were cultured with G-CSF to induce their differentiation either in the presence or absence of the MNK1-Inhibitor. Differentiation was assessed by morphology (Figure 5a, b) and CD11b surface antigen expression (Figure 5c, d). The percentage of antigen-positive cells and their fluorescence intensity were evaluated after 8 days of culture in the presence of G-CSF. G-CSF alone induced differentiation of up to 10% of 32D cells. Interestingly, the MNK1 inhibitor significantly enhanced differentiation of 32D in the presence of G-CSF. Up to 30% of 32D cells were differentiated upon exposure to CGP57380 and G-CSF (Figure 5c, d).

Figure 5
figure5

MNK1 inhibition enhances differentiation in 32D and HL60 cells. 32D cells were induced to differentiate towards granulocytes by 30 ng/ml G-CSF. HL60 cells were treated with 10 ng/ml TPA to induce monocytic differentiation and with 1.3% DMSO to induce granulocytic differentiation. Experiments were performed either in the presence or absence of MNK1 inhibitor CGP57380 (5 or 10 μ M). Cells exposed to the inhibitor showed enhanced differentiation as assessed by morphology (32D cells) and flow cytometry for CD11b expression. (a) Morphology of 32D cells in the absence of CGP57380. (b) Morphology of 32D cells in the presence of 10 μ M CGP57380. (c, d) Differences in differentiation status of 32D cells were confirmed by flow cytometry analysis of CD11b surface antigen expression. (e, f) Monocytic differentiation of HL60 cells with TPA was increased in the presence of either 5 or 10 μ M CGP57380 as verified by CD11b expression. (g) DMSO-induced granulocytic differentiation was also enhanced by CGP57380 (10 μ M)

These data indicate that MNK1 inhibition enhanced the ability of 32D cells to differentiate in response to G-CSF stimulation.

Differentiation experiments were also performed with HL60 cells. These cells can be differentiated towards granulocytes by DMSO exposure and towards monocytes by TPA. The MNK1 inhibitor significantly enhanced differentiation compared to cells exposed to TPA or DMSO alone (Figure 5e–g). Thus, MNK1 inactivation stimulates myeloid differentiation in several cell line models.

MNK1 expression by immunohistochemistry

The results that MNK1 downregulation or inactivation was associated with myeloid differentiation suggested that high MNK1 levels might play a role in leukemia. Since MNK1 was mainly regulated at the protein level, we used immunohistochemistry to analyse MNK1 expression in AML patients. Bone marrow biopsies from 112 patients including 89 patients with an initial diagnosis of AML were prepared as tissue arrays and analysed for MNK1 protein expression (Figure 6a–e). MNK1 was expressed with cytoplasmic localization in 25 of 99 samples. MNK1 was most prominently expressed in samples from patients with FAB M2 and M3 morphology (Figure 6f). These subtypes are often (AML1-ETO – FAB M2) or almost always (PML-RARα – FAB M3) associated with balanced translocations. No MNK1 expression was found in patients with erythroleukemia. The c-myc oncogene is another protein known to act on protein translation, and is also induced by several AML fusion proteins (Muller-Tidow et al., 2004b). The c-myc expression is also regulated by translational control. Therefore, we compared c-myc expression in the AML samples with MNK1 expression. The c-myc protein was expressed at varying levels in 70% of the AML patients. Patients with high levels of c-myc (Figure 6g) more frequently expressed MNK1 compared to those patient samples with absent or low c-myc expression (P=0.003, χ2 test).

Figure 6
figure6

MNK1 protein expression in bone marrow biopsies from AML patients. Immunohistochemistry with anti-MNK1 antibody (Cell Signaling) was used to analyse MNK1 protein expression in 112 bone marrow biopsies. (a) Picture of the entire array. Examples of AML samples, which express (b, d) or do not express (c, e) MNK1 protein, are shown at two magnifications: low (b, c) and high (d, e). (f) Percentage of MNK1-positive AML specimens in relation to their FAB subtype. (g) Association between c-myc expression levels and MNK1 expression. AML samples were analysed for c-myc immunoreactivity as described (Müller-Tidow et al., 2004 #1062). Samples were regarded on a scale of 0, +, ++ and +++. The bars indicate the percentages of MNK1-positive samples in each group. Statistical analysis (χ2 test) demonstrated an association between high levels of c-myc and increased MNK1 expression (P=0.03)

Discussion

Current models for the pathogenesis of acute myeloid leukemia favor a two-hit pathogenesis. In this model, common balanced translocation like PML-RARα, PLZF-RARα and AML1-ETO contribute to leukemogenesis by inducing a block in differentiation and by enhancing self-renewal. The action of these fusion proteins is thought to occur mainly at the transcriptional level. In the current study, we demonstrate that AML fusion proteins induce MNK1 protein expression. This finding is remarkable for several reasons. First, the regulation of MNK1 occurs at the protein level, followed by a decrease of the mRNA levels. Second, and even more importantly, this is the first time that AML fusion proteins were linked to oncogenic signaling on the level of protein translation. Finally, functional analyses revealed that MNK1 plays an important role in differentiation and proliferation of hematopoietic cells.

We recently performed microarray analyses to identify target genes that are shared by several AML fusion proteins (Muller-Tidow et al., 2004b). This approach is based on the assumption that shared target genes bear a high potential to be of functional relevance for leukemic pathogenesis. Overall, 63 common target genes of PML-RARα, PLZF-RARα and AML1-ETO were identified. Several of these genes play a role in transcriptional control. In addition, we deciphered the Wnt-signaling pathway to be induced by the fusion proteins. However, the function of several identified target genes remained unknown. We decided to study MNK1, a serine threonine kinase, in more detail since MNK1 regulates the eIF4E oncogene (Waskiewicz et al., 1999). The microarray analyses indicated that MNK1 mRNA was decreased by about 50% following expression of either AML1-ETO, PML-RARα or PLZF-RARα in U937 cells. These findings were confirmed by real-time RT–PCR for PML-RARα and PLZF-RARα, whereas no significant changes were found for AML1-ETO.

When we performed Western blot analysis, MNK1 levels were significantly increased by the AML fusion proteins. These experiments were repeated several times with different cell lysates and consistently showed induction by the fusion proteins. Also, MNK1 induction was found in tet-inducible U937-AML1-ETO cells, indicating that MNK1 expression was independent from zinc induction. These findings indicated that MNK1 induction occurred at the protein level and further analyses showed that PML-RARα enhanced MNK1 protein stability. Consequently, the exposure of the PML-RARα-expressing NB4 cells to ATRA reduced MNK1 protein levels in a dose-dependent manner. These findings indicate that MNK1 is regulated by PML-RARα at the protein level. One possible explanation for the observed downregulation of MNK1 mRNA is a secondary effect, most likely as a negative feedback loop.

To identify the mechanism leading to MNK1 overexpression, we performed protein half-life studies. MNK1 protein degradation showed a biphasic course with relatively high stability for the first hours and rapid degradation afterwards. PML-RARα prolonged the MNK1 half-life especially when protein synthesis was arrested for several hours. In line with these findings, ATRA reduced MNK1 protein levels and half-life in naturally PML-RARα-positive NB4 cells. These findings suggest that MNK1 overexpression results at least partially from increased stability. Other, so far unidentified mechanisms may also contribute to MNK1 overexpression.

MNK1 is a mitogen and stress-activated MAPK-activated protein kinase. MNK1 phosphorylates the eucaryotic initiation factor 4E (eIF4E) on Ser209. We demonstrated widespread MNK1 expression in leukemic cells. Overexpression of MNK1 in 32D cells enhanced eIF4E phosphorylation, which could be decreased by the MNK1 inhibitor CGP57380 and thus indicated the phosphorylation of eIF4E by MNK1. The eIF4E protein, a subunit of the eucaryotic cap-binding protein complex, specifically recognizes and interacts with the cap structure of mRNA. EIF4E activation by MNK1-based phosphorylation enhances translation of mRNAs with complex 5′UTR structure (Lazaris-Karatzas et al., 1990; Sonenberg and Gingras, 1998). One gene, implicated in the translational changes after MNK1 activation is the RANTES factor of late activated T cells (RFLAT-1) (Nikolcheva et al., 2002). However even in this case, it is not clear whether this is a direct effect of MNK1. No other genes have been identified so far, whose translation is controlled by MNK1. However, several lines of evidence indicate the importance of eIF4E and its potential importance for growth and proliferation. Transgenic Drosophila expressing a nonphosphorylatable form of eIF4E in an eIF4E mutant background had reduced viability, smaller siblings and developmental delays (Lachance et al., 2002). Furthermore, the tightly regulated activity of eIF4E (Gingras et al., 1999) is critical to normal cell growth (Flynn and Proud, 1996) and an antiapoptotic role for eIF4E has been demonstrated (Tan et al., 2000). Dysregulation or overexpression of eIF4E can lead to oncogenic transformation (Lazaris-Karatzas et al., 1990; Rousseau et al., 1996; Topisirovic et al., 2002). Elevated eIF4E levels were found in a broad spectrum of transformed cell lines and solid tumors (Miyagi et al., 1995). These and several other studies implicate eIF4E in the pathogenesis of human cancers (Dua et al., 2001). Recently, eIF4E was reported to take part in the formation of PML-containing nuclear bodies (Topisirovic et al., 2003a) and in cyclin D1 nucleocytoplasmic transport. The eIF4E protein also interacts with and is inhibited by the proline-rich homeodomain (PRH) protein in myeloid cells (Topisirovic et al., 2003a). The localization of eIF4E to PML-containing nuclear bodies provides another link to PML-RARα that disrupts these bodies (Topisirovic et al., 2003a). All these findings implicate eIF4E in growth control and proliferation. So far, the physiological functions of MNK1 phosphorylating eIF4E are incompletely understood and little is known about the role of MNK1 in cancer.

Our finding that MNK1 protein is induced by several AML fusion proteins hinted to an involvement in hematopoietic proliferation and differentiation. Therefore, we analysed the potential functions of MNK1 in hematopoietic cells. Expression analyses indicated that MNK1 protein levels decreased upon induction of myeloid differentiation. On the functional level, MNK1 decreased and slowed myeloid differentiation, since MNK1 inhibition enhanced myeloid cell differentiation. Also, MNK1 function is likely to be necessary for myeloid cell proliferation: Mutated MNK1 proteins were severely inhibiting the formation of colonies and no stable cell lines could be established from MNK1 mutant-transfected cells. In immunohistochemistry studies, we detected MNK1 expression in a significant percentage of AML patients. Interestingly, MNK1 expression was associated with high levels of c-myc protein expression. Since c-myc protein expression is tightly controlled at the translational level, the involvement of MNK1 in c-myc protein expression warrants further studies. It is likely that the role of protein translation and its regulatory mechanisms play a more important role in hematopoiesis and leukemia pathogenesis than currently anticipated. Further analyses of these mechanisms might lead to the development of novel targets for more specific therapies.

In conclusion, we demonstrate induction of the MNK1 protein by several AML fusion proteins. The translation regulating MNK1 plays an important role in hematopoietic proliferation and its inhibition facilitates differentiation of hematopoietic progenitor cells.

Materials and methods

Quantitative real-time RT–PCR

Total RNA was isolated using TRIZOL reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's recommendations. Total RNA (1 μg) was used for reverse transcription. The cDNA was diluted to 200 μl with ddH20, and 2.5 μl were used for each PCR reaction. The quantitation of mRNA levels was carried out using a real-time fluorescence detection method as described before (Linggi et al., 2002; Müller et al., 2000; Müller-Tidow et al., 2001a). Primers and probe: MNK1-forward primer: 5′-IndexTermIndexTermGAGAAGCCAGCCGAGTGGT-3′, MNK1-reverse primer: 5′-IndexTermIndexTermTGCCTTTGGTATGCAGGAAGT-3′ and MNK1-probe: 5′-IndexTermIndexTermCGGGACGTTGCTGCTGCCCTT-3′. Relative gene expression levels were calculated using standard curves generated by the serial dilutions of cDNA. All samples were independently analysed at least twice for each gene. The housekeeping gene GAPDH served as an additional control for the cDNA quality.

Cell lines and experimental design

U937, NB4, HL60, KCL-22, ML-1 and 32D cells were cultured in RPMI with L-glutamine and 10% fetal calf serum FCS. For 32D cells, WEHI supernatant was added as a source of IL-3. The stably transfected U937 cell lines that express PML-RARα, PLZF-RARα AML1-ETO or PMT-empty vector control in a Zn2+-inducible fashion have been previously described (Grignani et al., 1993; Ruthardt et al., 1997; Ferrara et al., 2001). Briefly, for induction of the fusion genes in these cells, 0.1 mM ZnSO4 was added to the culture media for 24 h. After induction, cells were either harvested for cell lysates or RNA preparation or used for cell cycle analyses.

HL60 cells were exposed to different concentrations of rapamycin (CalBiochem, CA, USA) for 24 h. Cell lysates were prepared using radioimmunoprecipitation buffer (RIPA) and Western blot experiments for MNK1 and actin were performed.

32D cells were treated with 5 μ M CGP57380 (MNK1 inhibitor, Novartis) for different time points (Knauf et al., 2001). After preparation of cell lysates, Western blot analyses were performed for eIF4E, phospho-eIF4E, MNK1 and actin.

Antibodies and Western blot analyses

Cell lysates preparation and Western blotting were performed as described (Müller-Tidow et al., 2001b). Briefly, cells were washed once in ice-cold PBS and lyzed for 30 min on ice in RIPA containing 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS 50 mM Tris (pH 8.0) with proteinase inhibitors (Complete; Boehringer Mannheim, Germany) and 1 mM sodium orthovanadate. Debris was removed by centrifugation at 20 000 g for 15 min. After adjustment of protein concentrations, the lysates were boiled in SDS sample buffer for 5 min and separated by SDS–PAGE. Gels were blotted on a PVDF membrane (Immobilon P, Millipore, Bedford, MA, USA) and stained with the indicated antibody. Antibody binding was detected with a horseradish-peroxidase (HRP)-coupled secondary antibody followed by chemoluminescence detection (ECL Plus, Amersham Pharmacia, Upsala, Sweden).

Antibodies against eIF4E and phospho-eIF4E were obtained from Santa Cruz (Santa Cruz, USA). The MNK1 antibody was purchased from Cell Signaling (Beverly, USA). HRP-coupled goat anti-rabbit and goat anti-mouse antibodies were purchased from Jackson Immuno Laboratories (West Grove, PA, USA). The antiactin mouse monoclonal antibody was obtained from Sigma (Taufkirchen, Germany).

MNK1 protein half-life analyses in fusion protein-expressing cells

U937 and U937-PML-RARα cells (3 × 105 cells/ml) were zinc induced (final concentration 0.1 mM). Cycloheximide was added after 24 h at a final concentration of 50 μg/ml. Cells were harvested at the indicated time points in RIPA buffer and Western blotting was performed. For densitometry analyses, the Blots were developed by using an INTAS camera (Epichem3 Darkroom) and the programme GelPro Analyser (1D-Gel ToolBar).

ATRA exposure and MNK1 protein half-life in NB4 cells

A total of 2 × 105 NB4 cells were seeded and incubated either with ATRA (10−6M) or ethanol as control. Cycloheximide (50 μg/ml; Sigma, Taufkirchen, Germany) was added after 24 h at a final concentration of 50 μg/ml. Also, NB4 cells were exposed to ATRA at varying concentrations for 24 h. Cells were harvested at the indicated time points. Western blotting was performed as described (Müller-Tidow et al., 2001b).

Cell cycle analysis

U937 cells stably transfected with either PMT vector as a control, PML-RARα or AML1-ETO were harvested after 0.1 mM Zn2+ induction for 24 h. Cells were washed with PBS (1% BSA) and fixed in ice-cold 70% ethanol in PBS for 60 min on ice. After an additional washing step, cells were treated with RNase A (5 mg/ml) for 10 min before adding 50 μg/ml propidium iodide for at least 30 min. Cell cycle profiles were analysed by flow cytometry.

Colony growth assays of 32D cells transfected with MNK1 and generation of stable cell lines

The 32D cells were electroporated with either 20 μg of pcDNA3.1 or the different pcDNA3.1-MNK1-expression vectors: MNK1-WT, MNK1-MA or MNK1-AA (kind gift from Dr H Gram, Novartis Pharma AG, Basel). The kinase-deficient mutant MNK1 (MA) and the phosphorylation site mutant (AA) of human MNK1 were described (Knauf et al., 2001).

The day after electroporation, cells were seeded in triplicate in methylcellulose colony assays (Mizuki et al., 2000) in the presence of neomycin (concentration 0.5 mg/ml) as a selection marker. Colonies were counted after 10 days. To generate stable MNK1-expressing cell lines, colonies were picked and expanded in WEHI-containing RPMI medium. Data were evaluated using the nonparametric Mann–Whitney U-test. All tests were performed two-sided and a P<0.05 was considered to be significant.

Differentiation of myeloid leukemia cell lines

32D cells (2 × 105/ml) were seeded in 4 ml RPMI medium, containing L-glutamine, sodium pyruvate and 10% FCS in the presence of granulocyte colony-stimulating factor (G-CSF; 30 ng/ml). G-CSF was freshly added every 48 h. When indicated, the cells were also exposed to 10 μ M CGP57380. CGP57380, a novel low-molecular-weight MNK1/2 kinase inhibitor, was kindly provided by Novartis (Tschopp et al., 2000). After 8 days, cells were washed with PBS, resuspended in 100 μl PBS and incubated with the (PE-labeled) anti-CD 11b antibody (clone M1/70.15.1, Cymbus Biotechnology) for 30 min on ice. Cells were washed once and kept on ice until FACS analysis.

For TPA-induced monocytic differentiation or DMSO induction of granulocytic differentiation, HL60 cells were plated in RPMI medium (containing 10% FCS) at 3 × 105/ml density. Cells were grown either in the presence or absence of 10 ng/ml TPA or 1.3% DMSO and harvested at the indicated time points. Additionally, cells were exposed to either 5/10 μ M CGP57380 or carrier. HL60 differentiation was confirmed by FACS analysis with (PE-labeled) anti-CD 11b antibody and anti-CD14 antibody (FITC-labeled). Western blot experiments were carried out using phospho-eIF4E, eIF4E, MNK1 or actin.

Tissue array construction and immunohistochemistry analyses

Tissue array construction of formalin-fixed and paraffin-embedded trephine bone marrow biopsies of 85 patients diagnosed with primary, untreated AML was performed as previously described (Kononen et al., 1998; Muller-Tidow et al., 2004a). A diagnostic Giemsa-stained section served as control to enable the definition of areas with the highest amount of blast cells. Two cores per patient were arrayed to analyse intratumoral heterogeneity of MNK1 expression.

Tissue sections were mounted on SuperFrost/Plus slides and dewaxed in xylene. For MNK1 detection, the sections were autoclaved in 10 mM citrate buffer pH 6.0 (10 min, 121°C). After washing in PBS, sections were incubated with the primary antibody (MNK1, Cell Signaling, dilution 1 : 20). The D-APAAP method was used for detection. MNK1 expression was regarded as negative or positive.

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Acknowledgements

We are grateful to Marion Baas for excellent technical assistance, to Dr Dong-Er Zhang for tetracycline inducible U937-AML1-ETO cells, to Dr Hermann Gram (Novartis, Basel) for helpful comments, plasmids and the MNK1/2 inhibitor and to Annette Becker for carefully reading the manuscript. This work is supported by grants from the Deutsche Forschungsgemeinschaft (Mu 1328/2-3, Se 600/2-4, Mu 1328/3-7, SFB 293-A15), the José-Carreras Leukemia Foundation (R03/19f) and the IZKF (Ser2/041/04; Mu 12/096/04) at the University of Münster.

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Correspondence to Hubert Serve or Carsten Müller-Tidow.

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Keywords

  • AML
  • balanced translocation
  • myeloid differentiation
  • MNK1
  • eIF4E
  • c-myc

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