Introduction

Flt-3 (Fms-like tyrosine kinase-3) is a receptor tyrosine kinase expressed by hematopoietic progenitor cells. Its activity is essential for normal blood development and establishment of the immune system.¹ Mutations of the Flt3 gene have been reported in patients with acute myeloid leukemia (AML). The majority of these patients harbor internal tandem duplications (ITD) in the sequence encoding the juxtamembrane domain of the Flt3 receptor. These ITD mutations occur in approximately 30% of patients with AML.¹ We and others have shown that Flt3-ITD mutations induce transformation of myeloid cells in vitro and in vivo and Flt3-ITD mutations activate signaling pathways that are very similar to interleukin (IL)-3-induced signaling events.^{2, 3, 4} The serine-threonine kinase Pim2 is transcriptionally induced by both Flt3-ITD and IL-3.^{4, 5, 6} The second type of Flt3 mutation, reported in about 7% of AML patients, is an activating point mutation in the tyrosine kinase domain of the receptor (TKD) and often involves mutation of an aspartate to a tyrosine residue at position 835 (D835Y).¹ These mutations result in ligand-independent activation of the receptor.⁷ Recently, we and others have described differences in the signaling properties of Flt3-ITD and Flt3-TKD mutations in liquid culture.^{8, 9} While both mutations confer growth factor independence to myeloid cells, only Flt3-ITD is capable of transforming these cells as demonstrated by clonogenic assays. Thus, TKD mutations behave similarly to Flt3 wild-type receptor (Flt3-Wt) in the presence of its Flt3 ligand (FL).^{2, 8}

The Pim family of proto-oncogenes (Pim1, Pim2 and Pim3) encodes serine threonine kinases that are widely expressed in the hematopoietic system.¹⁰ Transcription of Pim1 and Pim2 is induced by a wide variety of growth factors via STAT5 activation.^{6, 11, 12} Pim1 and Pim2 are shown to protect hematopoietic cells from cell death, and deficiency of Pim kinases leads to reduced body size in knockout mouse studies.^{10, 13, 14, 15, 16, 17} Interestingly, when primary bone marrow-derived blast cells from patients with AML were assessed for Pim2 expression using quantitative RT-PCR, many AML patients showed overexpression of Pim2 regardless of the presence of Flt3-ITD mutations.² This suggests that Pim2 expression may be increased by mechanisms other than Flt3-ITD and points to a role for Pim2 in the pathogenesis of AML.

While Pim2 is important for Flt3-ITD-mediated transformation of 32D cells, Flt3-D835Y-activating point mutations or FL-stimulated Flt3 wild-type receptor do not induce Pim2 expression and are not sufficient to support transformation of myeloid cells.^{4, 8} Therefore, we hypothesized that Pim2 coexpression in 32D cells expressing Flt3-Wt or Flt3-TKD can induce transformation of these cells.

Here we show that Pim2 overexpression in 32D Flt3-Wt cells enhances proliferation by enhancing S-phase entry and induces transformation by synergizing with Flt3.

Materials and methods

Reagents

Recombinant human FL and recombinant murine IL-3 were purchased from PeproTech (Rocky Hill, NJ, USA). The antibodies for anti-Pim2, anti-CCAAT/enhancer-binding protein- (C/EBP), anti-PU.1, anti-STAT5a/b from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-phospho STAT5a/b antibody from Upstate Biotechnology (Lake Placid, NY, USA) and the mouse monoclonal anti-actin antibody were purchased from Sigma (Taufkirchen, Germany). Anti-BrdU FITC-conjugated antibody kit was purchased from Becton and Dickinson (NJ, USA).

Cell lines

The IL-3-dependent murine myeloid cell line 32Dcl3 (kindly provided by Dr Felicitas Rosenthal, Freiburg, Germany) was cultured in RPMI 1640 supplemented with 10% Walter and Eliza Hall Institute-3 (WEHI3 cell)-conditioned medium as a source of IL-3, 10% fetal calf serum (FCS), 1% penicillin/streptomycin and 1% L-glutamine. Cells were cultured in a humidified incubator at 37 °C with 5% CO₂.

Generation of stable 32D cell lines

Stable cell lines (32D-Flt3-Wt or Flt3-D835Y) were generated essentially as described.⁸ 32D-Flt3-Wt or Flt3-D835Y cells overexpressing Pim2 were generated by transduction with retroviral supernatants collected from Phoenix packaging cells transfected with Pinco-Pim2 or Pinco empty vector control.¹⁸ green fluorescent protein (GFP)-positive cells were sorted by flow cytometry. Pim2 expression was analyzed in these stable cell lines by western blotting.

Western blot analyses

Western blots were performed as described before.¹⁹ Immunoblots were incubated with antibodies against Pim2, C/EBP, PU.1 pSTAT-5, tSTAT-5, SOCS 1 or actin (Sigma) followed by incubation with secondary immunoglobulin G antibody conjugated with horseradish peroxidase.

³H-thymidine incorporation

A total of 2 10⁴ 32D Flt3-Wt cells over expressing Pim2 were deprived of IL-3 and serum (0.5%. FCS) for 12 h in 200 l medium in 96-well plate. Subsequently, cells were placed in medium with 10% FCS and supplemented with the indicated concentrations of FL. After an 8-h incubation period at 37 °C, 1 Ci (0.037 MBq) ³H-thymidine was added to each well, and cells were incubated for additional 12 h. Cells were harvested onto glass fiber filters, and emission of bound DNA was analyzed in a scintillation counter.

Clonal growth in methylcellulose

Colony assays with stable cell lines were performed as described before.⁸ Colony assays after transient transfection were performed by electroporating and seeding these cells 24 h later in methylcellulose with 20% FCS and 2 ng ml^-1 IL-3 and G₄₁₈ (0.6 mg ml^-1). Colonies were counted between days 8 and 11.

Transduction of mouse hematopoietic progenitor cells and colony assays

Bone marrow cells were harvested and colony assays were performed as described earlier.²⁰ Cells were transduced with retroviral supernatants collected from Phoenix packaging cells transfected with Pinco-Pim2 or Pinco empty vector control.¹⁸ Transduced cells were cultured for 2 days and sorted for GFP-positive cells. One thousand GFP-positive sorted cells were seeded in methylcellulose supplemented with IL-3, stem cell factor and IL-6. After 10 days, colonies were collected from methylcellulose and replated (5000 cells per dish) on fresh methylcellulose medium with supplements mentioned above. Remaining cells from the colony assay were used for western blot to analyze the expression of Pim2.

For differentiation analyses GFP-positive cells were cultured in the presence or absence of granulocyte colony-stimulating factor (GCSF) (50 ng ml^-1). Smears were prepared and stained by Wright–Giemsa staining. Surface expression of CD11 b was analyzed by flow cytometry after staining with anti-CD11b-PE-conjugated antibody. The percentage of GFP and CD11b-PE double-positive cells were assessed.

Apoptosis assay

Briefly, 32D Flt3-Wt or D835Y cells expressing Pim2 or empty vector constructs were seeded in RPMI medium containing FCS alone or FL (20 ng ml^-1) in combination. Cells were incubated with PKC412 at 50 nM final concentration. At indicated time points cells were stained with annexin V-PE and 7ADD following the manufacturer's instructions (Immunotech, Marseille, France). Double-positive cells were analyzed by flow cytometry using a FACSCalibur cytometer (Becton Dickinson, NJ, USA).

Detection of BrdU incorporation into DNA-synthesizing cells

Cell cycle analyses were performed by BrdU incorporation assay with propidium iodide staining. Stable 32D-Flt3-Wt cells expressing Pim2 were IL-3 starved and serum deprived (0.5% FCS) for 12 h. Subsequently, cells were stimulated with FL (20 ng ml^-1) and harvested at different time points after stimulation. Cells were pulsed with BrdU (10 M) 1 h before each harvest. Next, cells were stained with FITC-conjugated anti-BrdU antibody following the manufacturer's instructions. Briefly, cells were fixed in ethanol followed by denaturation and antibody incubation. Isotype controls were used. Cells were washed and incubated with propidium iodide (50 g ml^-1) for 30 min in the dark. Subsequently, DNA content (PI) and DNA synthesis (FITC-BrdU) were assessed by flow cytometry using a FACSCalibur cytometer (Becton Dickinson).

siRNA for Pim2

To generate short hairpin RNAs (shRNA) for Pim2 RNAi, Pim2 siRNA sense and antisense (Eurogentec, Köln, Germany), oligos were annealed and cloned into p-super vector (Oligoengine, WA, USA). Sequences of oligos cloned into pSuper vector were AAGGCTTCATGCTGGTCCT—oligo1 sense, AGGACCAGCATGAAGCCTT—oligo1 antisense, AAGGATGAGAACATCCTGA—oligo2 sense and TCAGGATGTTCTCATCCTT—oligo2 antisense. Clones were confirmed by sequencing. 32D Flt3-ITD cells were electroporated with 16 g of Pim2 shRNA (1 and 2) together with pcDNA3 in a ratio of 5:1, in order to select the transfected cells with G₄₁₈ antibiotic in colony assays. After 24 h cells were washed once and used for colony assay in methylcellulose in the presence of G₄₁₈. Expression of Pim2 in these cells was analyzed by western blot analysis.

Results and discussion

Pim2 is required for Flt3-ITD-mediated cellular transformation

Recently, we identified genes that were differentially expressed in the myeloid progenitor cell line 32D upon expression of Flt3-ITD.⁴ Expression of Pim2 was strongly induced (43-fold) in the presence of Flt3-ITD compared to Flt3 wild-type receptor, and overexpression of a kinase-inactive Pim2 mutant significantly suppressed Flt3-ITD-induced colony growth.^{4, 21} To address the importance of Pim2 in Flt3-ITD-mediated transformation, we co-transfected 32D cells stably expressing Flt3-ITD with shRNA against murine Pim2 and a plasmid carrying a neomycin resistance gene. Colony assays were performed in the presence of G₄₁₈ to select for the transfected cells. The number of clonogenic cells was decreased threefold in cells that were transfected with shRNA against Pim2 as compared with control-transfected cells (P<0.01) (Figure 1a). Pim2 protein levels were reduced 12-fold in Pim2-shRNA-transfected cells compared to the control-transfected cells (Figures 1b and c).

Figure 1.

Pim2 is required for Flt3-ITD-mediated transformation. (a) Colony growth of 32D Flt3-ITD cells in methylcellulose was analyzed in the presence or absence of short hairpin RNAs (shRNA) against Pim2. Each bar indicates the mean colony number from triplicates from one representative experiment (means.d.). (b) Reduced expression of Pim2 in 32D Flt3-ITD cells transfected with shRNA against Pim2 or control vector by western blot. (c) Densitometric analyses show 12-fold reduction in protein level of Pim2 by shRNA. The bar diagram indicates the ratio of the integrated optical density of Pim2 and actin bands shown in (b). For densitometry we used INTAS camera (Epichem, Darkroom) and GelPro Analyzer (1D-GelToolbar). All experiments were performed at least two times yielding comparable results.

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The finding that inhibition of colony growth was not complete could be due to the fact that Pim2 protein levels were not entirely abolished. Alternatively, other Pim family members, such as Pim1, may have partially substituted for Pim2 to cause residual colony growth. In fact, Pim1 protein is expressed in 32D cells, but Pim2 shRNA did not affect Pim1 expression (data not shown). These data confirm and extend previous observations showing that a loss-of-function mutant of Pim2 or siRNA against Pim2 significantly inhibited colony growth of 32D-Flt3-ITD or Ba/F3-ITD cells, respectively.^{4, 21} We do not rule out the possibility that Pim1 might compensate the effect of Pim2 in ITD cells. However, Adam et al.²¹ have shown that Pim1 knockdown using siRNA or suppression of Pim1 activity by using a kinase dead mutant of Pim1 does not significantly inhibit the growth of transformed cell lines, whereas Pim2 alone significantly inhibits the growth of these cells. Here in the present report, we used shRNA to inhibit Pim2 expression and show that Flt3-ITD-mediated transformation is partially dependent on Pim2.^{4, 21} Further experiments are necessary to conclude if one member of the Pim family plays a dominant role over the other in Flt3-ITD-mediated transformation.

Pim2 overexpression synergizes with FL to stimulate proliferation

In order to analyze the potential of Pim2 to complement Flt3 wild-type receptor signaling, we established stable cell lines by retroviral transduction of 32D Flt3 wild-type receptor-expressing cells with Pim2. Expression of Pim2 in stable cells was analyzed in the absence of IL-3, since IL-3 can induce Pim2 expression. As shown in Figure 2a, Pim2 was expressed at high levels in the stable cells, whereas Pim2 protein was not detectable in cells transduced with vector alone. FL stimulation had no effect on Pim2 expression, and expression of the close homologue Pim1 was not altered in Pim2-overexpressing cells (data not shown).

Figure 2.

Pim2 promotes proliferation of 32D-Flt3-Wt cells in the presence of Flt3 ligand (FL). (a) Stable expression of Pim2 in 32D cells, as indicated by western blot analyses. (b) Pim2 enhances FL-dependent proliferation. The 32D-Flt3-WT cells were stably transfected with Pim2 or control plasmid as indicated. They were starved for 10 h in 0.5% fetal calf serum (FCS) and were subsequently exposed to the indicated concentrations of FL or left unstimulated. Proliferation was assessed by ³H thymidine incorporation. Pim2-overexpressing cells proliferated significantly more, when compared to control cells (^*P<0.05). The means.d. of three independent experiments is shown. (c) DNA synthesis and cell cycle analyses by BrdU incorporation were performed on 32DFlt3-Wt/control and 32DFlt3-Wt/Pim2 cells. Cells were starved in medium containing 0.5% serum for 10 h and subsequently stimulated with FL for the indicated times. BrdU was added to the cultures 1 h before cells were harvested. Cells were stained with anti-BrdU antibody and propidium iodide. The analyses were performed by flow cytometry. The data shown are representative of two independent experiments. Pim2-containing cell lines showed a higher fraction of cells in S-phase than control cells. (d) Percentages of cells in early S phase as analyzed from BrdU incorporation assay presented in (c). Flt3-Wt cells overexpressing Pim2 or kinase inactive mutant of Pim2 were stimulated with FL and analyzed for S-phase entry at different time points. Data presented here are means.d. of two independent experiments.

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32D cells are dependent on IL-3 for their growth, and expression of Flt3-Wt in these cells allows to substitute IL-3 by FL.^{2, 22} To analyze the effect of Pim2 overexpression on proliferation, we performed ³H thymidine assays on 32D-Flt3-WT cells in the absence or presence of FL. In the absence of growth factors, Pim2-overexpressing cells showed basal proliferation (data not shown), whereas control 32D-Flt3-WT cells that had been transfected with empty vector instead of Pim2 readily underwent apoptosis and did not proliferate. Interestingly, in the presence of increasing doses of FL, Pim2 overexpression consistently enhanced proliferation, when compared to vector control. Overall, Pim2 overexpression synergized with Flt3 signaling, while Pim2 alone only had minor effects on 32D cell proliferation (Figure 2b).

To assess the effects of Pim2 overexpression on cell cycle progression, we analyzed BrdU incorporation in response to FL stimulation at different time points in serum and IL-3 -deprived 32D-Flt3-Wt cells. Pim2 cells did not arrest entirely in G₁ phase of the cell cycle after 10 h of starvation, and a significantly higher fraction of the cells were in late S phase as compared to control cells (Figure 2c). Starvation time of 10 h may not be sufficient to arrest all 32DFlt3-Wt-Pim2-expressing cells in G₁ phase, whereas most of the 32DFlt3-Wt control cells were arrested in G₁ phase (Figure 2c).

After stimulation with FL, Flt3-Wt and Pim2-overexpressing Flt3-Wt cells rapidly entered S-phase (Figures 2c and d). However, a significantly higher fraction of Pim2-positive cells entered S-phase than control cells. The percentage of cells in S phase was consistently higher in Pim2-overexpressing cells over the whole time course as compared to control cells (Figures 2c and d). This required Pim2 kinase activity, since the kinase-inactive mutant of Pim2 was unable to stimulate S-phase entry and showed similar results as empty vector cells (Figure 2d). Pim proteins have been suggested to play an important role in growth and proliferation, and a stimulatory role for cell cycle progression has been demonstrated for Pim1.^{10, 13, 23, 24, 25, 26, 27} In the present paper, we provide evidence that Pim2 enhances S-phase entry. These results indicate that Pim2 functions as an active kinase in regulating cell cycle progression during G₁/S-phase transition. Involvement of Pim1 in the cell cycle as a positive regulator at G₂/M transition has been described before, where Pim1 phosphorylates and inhibits the activity of the cell cycle inhibitor C-TAK1.²⁸ Moreover, Pim1 was demonstrated to directly bind to, phosphorylate and activate CDC25A, a phosphatase, which positively regulates G₁/S-phase transition of the cell cycle.²⁷ Also, Pim1 led to phosphorylation and inactivation of the cell cycle inhibitor, p21^cip1/waf1, and it was shown to be involved in mitosis.^{29, 30} Our finding that Pim2 can enhance G₁/S-phase progression in cooperation with FL is intriguing and provides a mechanism of how Pim2 may confer a proliferative advantage to these cells. The search for potential cell cycle regulators whose activity is modified by Pim2 is warranted. Currently, we are analyzing the interacting partners of Pim2 by a systematic approach yeast-two-hybrid screen.

Pim2 confers resistance toward apoptosis

Next, we analyzed the antiapoptotic effects of Pim2 on 32D-Flt3-Wt or Flt3-D835Y cells. We compared the proportion of Flt3-Wt or Flt3-D835Y cells undergoing apoptosis after growth factor withdrawal in the presence or absence of Pim2 at several time points. In the absence of growth factors and Pim2, 50% of 32D-Flt3-Wt cells underwent apoptosis within 48 h. In contrast, Pim2 alone could rescue a significant fraction of cells under these conditions (Figure 3a). No significant apoptosis was observed in Flt3-D835Y cells independent of Pim2 expression under growth factor-deprived conditions, which is comparable to Flt3-ITD (Figure 3a). Furthermore, we compared the effects of tyrosine kinase inhibitor (PKC412) on apoptosis induction in Flt3-Wt and Flt3-D835Y cells in the presence or absence of Pim2. We incubated 32D Flt3-Wt or Flt3-D835Y cells containing either Pim2 or vector alone and Flt3-ITD cells with PKC412 (50 nM) and analyzed the extent of apoptosis at the indicated time points (Figure 3b). Similar to Flt3-ITD cells, Pim2-overexpressing 32D Flt3-Wt cells were more resistant to PKC412-induced apoptosis than 32D Flt3-Wt control cells. Similarly in the presence of Pim2, 70% of the Flt3-D835Y cells survived the treatment, whereas only 30% of Flt3-D835Y cells without Pim2 were rescued from PKC412-induced apoptosis. Addition of FL to PKC412 rescued the cells from apoptosis to some extent (Figure 3c). Similar findings were observed when apoptosis was induced by UV irradiation of cells in the presence or absence of FL (data not shown).

Figure 3.

Pim2 provides apoptosis resistance to 32D Fms-like tyrosine kinase-3 (Flt3)-Wt and 32DFlt3-D835Y cells. (a) Proportion of apoptotic cells in growth factor deprived medium, analyzed by annexin V/propidium iodide staining by flow cytometry. Growth factors were withdrawn from the medium of 32D Flt3-Wt or 32DFlt3-D835Y cells overexpressing Pim2. Cells were harvested at indicated time points to stain with annexin V and 7.AAD. The proportion of double-positive cells is plotted against the time after growth factor withdrawal. (b) Percent of apoptotic cells after treatment with tyrosine kinase inhibitor (PKC412) at 50 nM concentration in fetal calf serum (FCS) medium. (c) Percent of apoptotic cells after treatment with tyrosine kinase inhibitor (PKC412) at 50 nM concentration in medium containing FL. Apoptotic populations were analyzed at the indicated time points same as described in (a). Data presented here are means.d. of 2 independent experiments.

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Taken together, these data indicate that Pim2 alone induced significant antiapoptotic effects (Figure 3a) that were moderately enhanced by FL (Figure 3c) and strongly by Flt3-D835Y (Figures 3b and c), whereas, for inducing proliferation it cooperates with Flt3 receptor.

Pim2 induces transformation of Flt3 wild-type or Flt3-D835Y overexpressing 32D cells and of primary murine bone marrow cells

Having shown that Pim2 is necessary for Flt3-ITD-induced colony growth and that it can complement the antiapoptotic and proliferative signals of Flt3-WT, we investigated whether Pim2 also alters clonogenic growth of 32D-Flt3-WT cells. As shown previously, 32D-Flt3-WT cells were not able to form colonies even in the presence of FL.² Interestingly, Pim2 overexpression was sufficient to induce clonogenic growth of Flt3-Wt expressing cells in the presence of FL. Notably, to fully exert this function, Pim2 needed additional signals that could be provided by FL or IL-3, but not by serum alone (Figure 4a). Thus, our data strongly point to the notion that wild-type Flt3 and Pim2 provide overlapping yet distinct signals toward proliferation and survival that synergize to result in a signal toward leukemic transformation resembling that of Flt3-ITD. Our data indicate that wild-type Flt3 and Pim2 are largely redundant in providing antiapoptotic signals (Figure 3). Previously, we and others have shown that Flt3-dependent survival signals are primarily mediated by activation of the PI3K-AKT pathway.^{8, 31} Indeed, both Pim2 and Akt are known to phosphorylate the antiapoptotic protein BAD,^{15, 16} suggesting that both pathways impinge on apoptosis regulation through modification of the Bcl2 family of proteins. Overexpression of Pim2 did not enhance the activity of AKT in our experiments (data not shown), as has been described previously.¹⁵ More evidence for the hypothesis that Pim2 and AKT are components of overlapping but yet independent pathways comes from studies by Hammerman et al.³² using Pim1 and Pim2 knockout mice. These authors showed that Pim2 is required for cell growth and survival when the AKT pathway is pharmacologically inhibited by rapamycin.³² Likewise, overexpression of either Pim2 or AKT led to increased cell size and resistance against apoptosis. Interestingly, coexpression of both Pim2 and AKT transgenes in mice induced the formation of lethal lymphomas in all double-transgenic animals, while none of the Pim2 single-transgenic mice and only one out of ten AKT single-transgenic mice developed lymphoma.³² In our study, we show that Pim2 acts synergistically with Flt3-Wt to induce transformation. This could be due to stabilization of the effect of AKT on cell survival and proliferation via an independent but parallel pathway.

Figure 4.

Pim2 synergizes with Fms-like tyrosine kinase-3 (Flt3)-WT and Flt3-D835Y in the induction of clonogenic growth. (a) The 32D Flt3-Wt cells expressing Pim2 or vector control were plated in triplicates at a density of 1000 cells per dish. Indicated cytokines were added to the medium. Colonies were counted on days 10. Each bar represents the means.d. of a representative triplicate experiment. (b) Colony numbers (means.d.) of 32D Flt3-D835Y cells in the presence or absence of Pim2 and/or FL. (c) Overexpression of Pim2 in 32DFlt3-D835Y cells as indicated by western blot analyses. (d) Colony numbers (means.d.) of 32D Flt3-D835Y and 32D Flt3- internal tandem duplications (ITD) cells in the presence or absence of Pim2 in fetal calf serum (FCS) containing methylcellulose. Each bar represents the meanSD of a representative triplicate experiment. (e) Primary mouse bone marrow cells were retrovirally transduced with Pim2 or control vector. They were seeded in methylcellulose in the presence or absence of growth factors as indicated. Colonies were counted on day 10. Data shown here are the means.d. (triplicates) of one out of four experiments (^*P<0.01). (f) Expression of Pim2 in colonies from primary bone marrow cells as indicated by western Blot analyses.

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Expression of an activating point mutation of Flt3 receptor, D835Y, in myeloid cell lines and in primary bone marrow induced a significantly different phenotype than Flt3-ITD and, Flt3-ITD induces a myeloproliferative syndrome in a retroviral transplantation model of primary mouse bone marrow, while Flt3-D835Y did not.⁹ A good surrogate parameter for the differential transforming potential of these mutations was their ability to induce clonogenic growth of 32D cells in semisolid media. While Flt3-ITD-induced colony growth in 32D cells, Flt3-D835Y (like Flt3-WT in the presence of FL) did not.⁸ To investigate whether Pim2 can confer clonogenic growth properties to 32D-Flt3-D835Y cells, we established a stable cell line by retrovirally transducing Pim2 into 32D-Flt3-D835Y cells. When we analyzed the colony growth of these cells in semisolid media in the presence or absence of FL, we observed that Flt3-D835Y cells overexpressing Pim2 were now able to form colonies, regardless of the presence of FL (Figure 4b).⁸ Stable high expression of Pim2 was confirmed by western blot in these cells (Figure 4c). Obviously, Pim2 complements signals provided by Flt3-WT or Flt3-D835Y to resemble the transforming capacity of Flt3-ITD (Figure 4d).

In order to confirm our findings in primary cells, a bone marrow colony assay was performed. Pim2 expression significantly enhanced colony growth of primary mouse bone marrow as compared to control (P<0.01) (Figure 4e). Pim2 overexpression in these colonies was confirmed by western blotting (Figure 4f). We also seeded Pim2-transduced bone marrow cells in colony assays in the absence of growth factors to analyze whether Pim2 can provide growth factor independence to primary progenitor cells as shown for Flt3-ITD. However, we did not observe any colony growth under these conditions (Figure 4e). These data show that Pim2 alone is not able to confer growth factor independence. Pim2 did not alter the replating efficiency of colonies as compared to empty vector (data not shown).

Transforming potential of Pim2 is independent of differentiation

We wanted to further investigate whether the growth advantage induced by Pim2 in Flt3-Wt cells was associated with a disruption of myeloid differentiation and/or expression of C/EBP and PU.1 as has been described for Flt3-ITD.^{4, 33} Analyses of protein levels of these transcription factors in 32D Flt3-Wt cells with Pim2 overexpression showed no significant change in the expression of C/EBP and PU.1 compared to control cells regardless of the presence or absence of FL (Figure 5a). Likewise, overexpression of Pim2 did not inhibit GCSF-induced myeloid differentiation of primary murine bone marrow cells as assessed by morphology and CD11b surface marker expression (Figures 5b and c).

Figure 5.

Pim2 does not block differentiation of myeloid cells (a) Western Blots show that Pim2 does not effect the expression of transcription factors involved in differentiation. (b) Wright–Giemsa staining of bone marrow cells expressing Pim2 or empty vector control. Primary murine bone marrow cells transduced with Pim2 or empty vector after sorting for GFP-positive cells were exposed to granulocyte colony-stimulating factor (GCSF) at concentration of 50 ng ml^-1 for 8 days or left untreated. Upper panel shows unstimulated cells and lower panel shows GCSF-treated cells (c) Histograms showing the expression of CD11b analyzed by surface staining and flow cytometry. The black-filled curve represents the GCSF-treated cells, the empty curve shows the untreated cells. (d) Pim2 does not enhance STAT5 phosphorylation in Fms-like tyrosine kinase-3 (Flt3)-D835Y cells. Western blots showing STAT5 phosphorylation and SOCS1 expression in 32D cells expressing indicated constructs. Cells were starved for 10 h in medium containing 0.5% fetal calf serum (FCS), followed by overnight incubation with Flt3 ligand (FL) or interleukin (IL)-3. Total cell lysates were separated by SDS–PAGE and blots were probed with antibody specific for phospho STAT5 (Y694/699). Blots were striped and reprobed with total STAT5 antibody followed by SOCS1 and actin antibodies.

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Thus, it appears that while Pim2 may confer many of the growth-stimulatory effects of Flt3-ITD, other pathways are responsible for the Flt3-ITD-mediated differentiation block. It will be interesting to dissect these pathways and identify further specific targets of Flt3-ITD that may enhance the differentiation block in patients with AML.

Transforming potential of Pim2 in D835Y cells is independent of STAT5 activation

We observed strong antiapoptotic and transforming effects of Flt3-D835Y cells in the presence of Pim2. As described previously, Flt3-D835Y induces very weak activation of STAT5.⁸ We analyzed whether Pim2 induces STAT5 activation in these cells. We compared STAT5 activation in Flt3-D835Y cells expressing Pim2 or empty vector alone in the presence of the indicated growth factors (Figure 5d). STAT5 was very weakly activated in D835Y cells in the presence of FL or FCS alone compared to IL-3 (Figure 5d). Pim2 overexpression in D835Y cells did not enhance STAT5 activation in all three conditions, while Flt3-ITD cells harbored strong STAT5 phosphorylation regardless of Pim2 expression (Figure 5d). We also analyzed SOCS1 expression levels in Pim2-overexpressing 32D-D835Y cells since Pim and SOCS family proteins function as components of negative feedback mechanism to regulate STAT5 activity.³⁴ Pim2-mediated phosphorylation of SOCS1 would be expected to stabilize SOCS1 levels in the cell.³⁵ Here no change in the SOCS1 levels were observed in response to Pim2 overexpression in 32D-Flt3 D835Y cells (Figure 5d). These data suggest that Pim2-induced transformation of 32D D835Y cells is independent of its influence on the upstream SOCS1/STAT5 pathway. Rather, alternative pathways such as altered cell cycle regulation or inhibition of apoptosis by Pim2 could be responsible for this transformation.

Taken together, our findings suggest that Pim2 and Flt3 act through different but complementary pathways to stimulate cell cycle progression and inhibit apoptosis. Together, they can transform hematopoietic progenitor cells similar to Flt3-ITD. Thus, Pim2 may be an interesting target for novel specific antileukemic therapies.

Notes

Authors' contribution SA, CMT and HS designed the study and SA performed most of the experiments and analyzed the data. SA, SK, HS wrote the manuscript. NB, NGR, WB, HS and CMT provided tools and analysis methods. All authors checked the final version of the manuscript.

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Acknowledgements

We thank Professor/Dr M Eilers (Institute for Molecular Biology and Tumor Research, University of Marburg, Marburg) for providing pSuper vector; C Choudhary for 32D-Flt3-TKD mutant cell lines; Dr LTickenbrock for helping with densitometric analysis and S Doths for technical assistance. This work is supported by the Deutsche Forschungsgemeinschaft (Se 600/3-1, SFB293), Thyssen-Stiftung (10.05.2.178), Deutsche Krebshilfe (10-2258,10-1539, 106697) and the Medical Faculty of the University of Münster (IZKF Ser2/041/04, IMF Sa 110404).