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Molecular Biology, Pathobiology and Genetics |
1 Department of Medicine, Hematology and Oncology; 2 Interdisciplinary Center for Clinical Research; and 3 Institute of Pathology, University of Münster, Münster, Germany and 4 Comprehensive Cancer Center and Cancer Research Institute, University of California, San Francisco, California
Requests for reprints: Christian Brandts, Department of Medicine, Hematology and Oncology, University of Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany. Phone: 49-251-83-52490; Fax: 49-251-83-52673; E-mail: cbrandts{at}uni-muenster.de.
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Abstract |
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Introduction |
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Activating Flt3 mutations have been reported in all AML subtypes and represent the most common molecular defect in de novo AML. Oncogenic N-Ras and K-Ras mutations are found in a quarter of all cases of AML but rarely occur together with Flt3-ITD (9), suggesting functional overlap.
Oncogenic Ras has been shown to activate the phosphatidylinositol 3-kinase (PI3K; ref. 10), which leads to accumulation of phosphatidylinositol (3–5) trisphosphate at the cell membrane and binding of the serine-threonine kinase Akt via its pleckstrin homology domain. At the cell membrane, Akt undergoes a conformational change and is phosphorylated on Thr308 by PDK1 and Ser478 by an unknown kinase, leading to Akt activation (11). Akt has been shown to mediate survival and proliferation, partly through inhibition of Foxo transcription factors (11).
Foxo3a is a member of the Foxo subfamily of forkhead transcription factors that plays a critical role in apoptosis and cell cycle regulation. The three members of the forkhead family, Foxo1 (FKHR), Foxo3a (FKHRL1), and Foxo4 (AFX) represent mammalian orthologues of a Caenorhabditis elegans transcription factor, DAF16. Constitutively active Akt and Akt activated in response to growth factor stimulation phosphorylates Foxo3a. Through binding to 14-3-3 scaffolding proteins, Foxo3a is retained in the cytoplasm and its transcriptional activity is prevented (12, 13).
We and others (14, 15) have found that expression of Flt3-ITD in myeloid 32Dcl3 cells leads to interleukin-3 (IL-3)–independent survival and proliferation. Furthermore, constitutive signaling via the signal transducers and activators of transcription 5 (STAT5) transcription factor has been observed (14, 16, 17). However, the mechanisms of Flt3-ITD–mediated transformation are still only partially understood.
Given the emerging importance of the PI3K/Akt/Foxo pathway, we were interested to determine its relevance in Flt3-ITD–mediated transformation of myeloid cells. Here, we provide evidence that Flt3-ITD induced constitutive Akt activation and, in turn, inactivated its substrate Foxo3a. Conditionally active Akt mediated survival, proliferation, and leukemic transformation of myeloid cells both in vitro and in vivo, likely through inactivation of Foxo3a transcriptional activity. Our data provides a potential mechanism of Flt3-ITD–mediated transformation.
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Materials and Methods |
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The generation of 32D cells stably transfected with wild-type Flt3 or Flt3-ITD has been previously described (14, 18). Briefly, the full-length coding sequence of Flt3 from a patient with ITD mutation as well as the full-length wild-type sequence from the Oci-AML5 cell line were amplified, cloned into the pAL expression vector, and stably transfected into 32D cells (14, 18). The expression of these receptors was regularly measured by immunoblotting and fluorescence-activated cell sorting (FACS).
We generated 32D cells stably expressing a conditionally active form of Akt as previously described (19, 20). In brief, the pleckstrin homology domain of Akt was replaced by the c-Src myristylation-targeting sequence (M+, hereafter referred to as M+Akt), leading to PI3K-independent membrane localization. Conditionality was conferred by fusing M+Akt to a modified form of the mouse estrogen receptor (ER) that binds 4-hydroxytamoxifen (4-OHT) but is refractory to estrogen (ER*). Addition of 4-OHT competitively removes sequestered heat shock proteins and activates the fusion protein (21). As a control, we expressed a myristylation-defective form of the protein (M–Akt:ER*), which is not membrane associated and therefore inactive (20). Both 32D-M–Akt:ER* and conditional 32D-M+Akt:ER* cell lines used for experiments were pooled populations.
A mutant form of Akt that contains alanine substitutions at residues Ser473 and Thr308 was kindly provided by Dr. D. Stokoe (22). To delete all kinase activity, we substituted K179 with alanine by site-directed mutagenesis, thereby creating a dominant-negative Akt/K179A-T308A-S473A mutant (referred to as dnAkt).
A mutant form of Foxo3a that contains alanine substitutions at three phosphorylation sites can neither be phosphorylated nor sequestered in the cytosol when Akt is activated (12, 13, 23). We generated 32D cells stably transfected with pcDNA-Foxo3a/A3:ER*, a plasmid which was kindly provided by Dr. P. Coffer (23). The phosphorylation sites Thr32, Ser253, and Ser315 are mutated to alanine, and Foxo3a/A3 is fused to ER*. Thereby, Foxo3a can be activated by addition of 4-OHT. 32D cells coexpressing Flt3-ITD and Foxo3a/A3:ER* were pooled populations.
Immunoblot Analysis and Antibodies
Cells were lysed in buffer containing 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.5), 1% NP40, 1 mmol/L EDTA, 1 mmol/L EGTA, 25 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L DTT, and protease inhibitor cocktail (Complete mini; Boehringer, Mannheim, Germany) at 4°C. Lysate was centrifuged (10,000 x g) to remove insoluble components. Equal amounts of protein were loaded onto polyacrylamide gels and run by standard SDS-PAGE. Proteins were transferred to Immobilon-P membrane (Millipore, Bedford, MA) and blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 (PBS-T). Primary antibodies were anti-phospho-Ser473-Akt, anti-phospho-Foxo, (total) Akt, (total) Foxo (purchased from Cell Signaling Technology, Inc., Beverly, MA), and ß-actin (Sigma, Deisenhofen, Germany). This was followed by incubation with horseradish peroxidase–conjugated secondary antibodies (obtained from Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Proteins were detected with enhanced chemiluminescence (Amersham Pharmacia Biotech) and exposed to film.
Luciferase Reporter Assays
To determine Foxo transcriptional activity, we used the firefly luciferase reporter construct pGL2-6xDBE (containing six Foxo3a binding sequences) kindly provided by Dr. P. Coffer (23). 32D cells expressing either wtFlt3, Flt3-ITD, M–Akt:ER, or M+Akt:ER were electroporated (300 V/975 µF) with 15 µg of pGL2-6xDBE or empty vector (pGL2) and cotransfected with 25 ng of a Renilla luciferase plasmid (pRL-TK). Luciferase activity was measured using the Dual Luciferase Reporter System (Promega, Madison, WI). The values were normalized for transfection efficiency to the respective values of Renilla luciferase. Each data point represents the mean ± SD of three independent experiments.
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium Cell Viability Assays
The CellTiter 96 Aqueous Proliferation Assay (Promega, Madison, WI) is a colorimetric method for determining cell viability in a 96-well format. Fifteen microliters of the substrate 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) are added to 100 µL of cell culture for 1 hour and MTS is metabolized into formazan by living cells. The quantity of formazan product as measured by the amount of 490-nm absorbance is proportional to the number of living cells in culture. Experiments were repeated at least thrice.
[3H]-thymidine Incorporation
A total of 4 x 104 cells per well were plated in 200-µL medium containing 0.5% FCS. Cells were stimulated with either 20 ng/mL Flt3 ligand or 2 ng/mL IL-3 or cultured without cytokines for 24 and 48 hours. Six hours before each time point, 1 µCi (0.037 MBq) of [3H]-thymidine was added. Genomic DNA of the cells was harvested on glass fiber filters, and ß-emission of the bound DNA was detected with a scintillation counter. Experiments were repeated at least thrice.
Flow Cytometry
Cell cycle analysis. 32D cells were washed in PBS containing 0.1% bovine serum albumin (PBS-B) and fixed in 70% ethanol at 4°C for 1 hour. The cells were resuspended in PBS-B and treated with 5 mg/mL RNase A for 10 minutes. Propidium iodide (50 µg/mL, Boehringer) was added, incubated for 30 minutes at room temperature, and analyzed by FACS.
Annexin V/propidium iodide staining. Cells were washed once with PBS, resuspended in 250-µL calcium-binding buffer containing propidium iodide (1.5 µg/mL) and Annexin V/FITC (0.05 µg/mL; purchased from Immunotech, Marseilles, France), and incubated on ice for 30 minutes. FACS analysis was done by FACSCalibur (BD, Heidelberg, Germany), using CellQuest software.
Colony Assays
Growth in semisolid methylcellulose was taken as being representative of in vitro leukemic transformation. We analyzed clonal growth of 1 x 104 32D cells per dish in 1 mL of culture mix containing Iscove's modified Dulbecco's medium (Life Technologies, Grand Island, NY), 1% methylcellulose, and 20% FCS. 32D cells expressing M–Akt:ER* or conditionally active M+Akt:ER* were treated with 2 µmol/L 4-OHT. The colonies were counted on day 8.
Animal Experiments
Nine-week-old female C3H/HeJ mice, which are syngeneic to 32Dcl3 cells, were used to assess the in vivo development of leukemia-like disease. 32D-Flt3-ITD cells or 32D-M+Akt:ER* cells (1 x 106) were injected into the tail vein. Mice injected with 32D-M+Akt:ER* were treated by daily injection of either tamoxifen (which is metabolized to 4-OHT in the liver) or solvent (peanut oil), as previously reported (24). Moribund animals were sacrificed. Femurs and organs were removed, formalin fixed, and paraffin embedded. The experimental protocols were reviewed and approved by the local Committee on Animal Experimentation.
Tissue Array and Primary Acute Myeloid Leukemia Samples
The tissue array construction of 90 patients diagnosed with AML was done as recently described (25). The tissue array contained samples from 66 patients diagnosed with primary, untreated AML, 19 patients with relapsed AML, and five patients with refractory AML. A diagnostic Giemsa-stained section served to enable the definition of areas with the highest amount of blast cells. Two punches were arrayed per patient to control for intratumoral heterogeneity (26). Tissue sections were mounted on SuperFrost/Plus slides and dewaxed in xylene. The phospho-Ser473-Akt (p-Akt) antibody was tested on PTEN-mutant U87 cells that had been similarly fixed and embedded. The tissue array section was microwaved in 10 mmol/L citrate buffer (pH 6.0, 10 minutes, 120°C). After washing in PBS, the section was incubated with the primary p-Akt antibody (purchased from Cell Signaling Technology) at 1:100 dilution. The D-APAAP method was used for detection and a semiquantitative scoring system ranging from 0% to 100% (in 10% increments) was used to detect blast staining, as well as the nuclear and membrane/cytosolic localization. Approval for the use of primary AML samples was granted by the local Ethics Committee.
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Results |
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Dominant-negative Akt reverses Flt3 internal tandem duplication–mediated interleukin-3 independence. The 32D cell line is an IL-3–dependent murine myeloid precursor cell line. Expression of Flt3-ITD in 32D cells confers IL-3 independence (14, 18). On the contrary, wtFlt3-expressing cells are dependent on either Flt3 ligand or IL-3 (14, 18). As shown in Fig. 2C, we expressed dominant-negative Akt (22) in 32D cells stably expressing Flt3-ITD and observed a 75% decrease in IL-3-independent viability. Interestingly, the effects of dnAkt were partly rescued by the addition of IL-3, which argues for a specific requirement of Akt in Flt3-ITD–mediated survival.
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Restoration of Foxo activity prevents Flt3 internal tandem duplication–mediated growth properties. To examine whether forced Foxo activity could overcome Flt3-ITD–mediated growth properties, we used a conditional Foxo3a construct (23). The expression plasmid contains phosphorylation-insensitive Foxo3a fused to the estrogen receptor (Foxo3a/A3:ER*) that responds to 4-OHT. We generated 32D cells expressing conditional Foxo3a/A3:ER* and either wtFlt3 or Flt3-ITD. Induction of phosphorylation-resistant and therefore noninhibitable Foxo3a/A3 in Flt3-ITD–expressing cells prevented cell viability after 24 hours (Fig. 1D). Upon Foxo3a activation, 32D-Flt3-ITD cells underwent apoptosis, irrespective of concomitant Flt3 ligand stimulation. Taken together, the data presented in Fig. 1 suggest a linear pathway, whereby expression of oncogenic Flt3-ITD constitutively activates Akt, which in turn phosphorylates Foxo3a and inactivates its transcriptional activity. Restoration of Flt3-ITD–resistant Foxo3a activity overcomes the Flt3-ITD–mediated growth advantages.
Conditional activation of Akt mediates Foxo inactivation and protects from cytokine withdrawal. We were interested to know whether the altered growth and survival properties conferred by Flt3-ITD were, at least in part, mediated by activation of Akt. To test this, we used a conditionally active form of Akt targeted to the cell membrane (M+Akt:ER*; ref. 20). The myristylation-defective and inactive form of the same construct (M–Akt:ER*) served as a control and we stably expressed both forms in separate pools of 32D cells.
As shown in Fig. 2A, the M–Akt:ER* and M+Akt:ER* forms were expressed at equal levels as measured by total Akt detection. Whereas exogenous Akt in M+Akt:ER*-expressing cells was rapidly activated within 15 minutes of 4-OHT treatment, the myristylation-defective M–Akt:ER* was not. Concomitantly, Foxo-dependent transcriptional activity (as measured by luciferase reporter activity) was dose dependently inhibited in 32D M+Akt:ER* cells, whereas it was unaffected in 32D M–Akt:ER* control cells after 4-OHT treatment (Fig. 2A).
IL-3 withdrawal of IL-3-dependent 32D cells resulted in near-complete cell death within 24 and 48 hours (Fig. 2B, open columns). Similarly, 4-OHT treatment of M–Akt:ER*-expressing cells (leading to mislocalized and inactive Akt) did not result in increased cell viability (Fig. 2B, left, black columns). In contrast, cells induced to express membrane-targeted and active Akt (M+Akt:ER*) survived the cytokine withdrawal (Fig. 2B, right, black columns).
In an additional experiment, we generated stable clones of M+Akt:ER*-expressing 32D cells. In response to 4-OHT, several clones induced Akt activity that was quantitatively very similar to the Akt activation of Flt3-ITD (Fig. 2C). At these induction levels, Akt activation mediated a strong factor independence (Fig. 2C, right). In conclusion, activation of Akt at levels that are comparable with Flt3-ITD induced activation levels protect 32D cells from cytokine withdrawal.
Conditional activation of Akt promotes survival, cell cycle progression, and proliferation. To distinguish between protection from apoptosis or increased proliferation of a subset of cells (Fig. 2B and C), we did cell cycle analyses of 32D M+Akt:ER* cells. When cultured in the presence of IL-3, the cells were alive (sub-G1 fraction of 1%) and rapidly dividing, with an S-phase population of 39% (Fig. 3A, left). After 24 hours of IL-3 withdrawal (Fig. 3A, middle), a large proportion of dead cells accumulated in the sub-G1 fraction (40%), whereas only 10% and 12% cells progressed to S and G2-M phase, respectively. Upon Akt activation (Fig. 3A, right), the sub-G1 population was markedly reduced (20%). The G0-G1 population was evenly distributed (40%, 37%, and 41%; Fig. 3A, from left to right). Interestingly, more Akt-expressing cells were found in the S phase (20%) and the G2-M phase (20%) of the cell cycle, suggesting that Akt increases cell cycle progression (Fig. 3A, middle).
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In addition, proliferation was measured by [3H]-thymidine incorporation. Upon Akt activation, DNA synthesis was increased in surviving cells (Fig. 3C, right). Taken together, the data document a combination of increased survival and increased proliferation of surviving cells upon Akt activation.
Conditional activation of Akt promotes transformation. Finally, transformation can be measured by growth in semisolid methylcellulose. As a positive control, the IL-3-dependent 32D cells were treated with IL-3 and formed colonies as previously observed (Fig. 3D; ref. 14). Importantly, colony formation was observed in 32D-M+Akt:ER* cells treated with 4-OHT to activate Akt, whereas 4-OHT-treated 32D-M–Akt:ER* cells formed no colonies (Fig. 3D). In addition, colonies from Akt-expressing cells seemed smaller in size than IL-3–treated colonies (data not shown), suggesting a weaker Akt-mediated proliferation when compared with IL-3. Nevertheless, Akt activation was sufficient to promote in vitro anchorage-independent growth and transformation of myeloid 32D cells.
Conditional activation of Akt in vivo promotes a myeloid leukemia-like disease. We have previously used a syngeneic model with C3H/HeJ mice to induce Flt3-ITD-dependent leukemia-like disease (14, 27). Because we were particularly interested to dissect Akt as an important pathway downstream of Flt3-ITD, we injected C3H/HeJ mice with either 32D-Flt3-ITD or 32D-M+Akt:ER* cells. I.p. injection with tamoxifen (which is metabolized to 4-OHT in the liver) of 32D-M+Akt:ER* carrying mice was done daily. The first mice started to die from a leukemia-like disease 33 days after injection. As can be seen from Fig. 4A, mice treated with tamoxifen were particularly prone to develop a leukemia-like disease (median survival, 42 days). Similarly, mice carrying Flt3-ITD rapidly developed a leukemia-like disease (median survival, 51 days). Only 2 of 10 mice injected with 32D-M+Akt:ER* but treated with solvent as control died within the 8 weeks of the experiment (Fig. 4A). Survival of the control mice differed significantly from Flt3-ITD or Akt-ER* + tamoxifen mice (P < 0.05, log-rank test), whereas the two latter did not differ significantly (P = 0.61, log-rank test). Gross anatomic examination showed hepatomegaly, splenomegaly, and lymphadenopathy. Histologic evaluation revealed that all the mice with activated Akt showed massive infiltration of p-Akt-positive blasts in the bone marrow (Fig. 4B), as well as in the liver and spleen (data not shown).
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Discussion |
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Because Akt activation has been shown to mediate survival and proliferation in several nonmyeloid cell systems (22, 23, 29, 30), we wondered about the biological consequences in myeloid precursor cells. We generated myeloid cells to conditionally activate Akt and target it to the cell membrane. As a control, we used a myristylation-defective form, which leads to mislocalization and cannot be activated by addition of 4-OHT (Fig. 2A). We showed that activated Akt targeted to the cell membrane protected myeloid 32D cells from cytokine withdrawal (Fig. 2B) and apoptosis (Fig. 3A and B). The observed effects were not due to unphysiologically high levels of Akt activity, as similar observations were made with individual clones titrated to Akt activity observed in Flt3-ITD (Fig. 2C).
Interestingly, Akt affected proliferation by allowing progression from the G1 into S and G2-M phase of the cell cycle (Fig. 3A), with increased DNA synthesis in the S phase (Fig. 3C). This proliferative advantage of surviving cells allowed for clonal growth in semisolid methycellulose (Fig. 3D). However, Akt-mediated colonies were smaller in size when compared with IL-3-treated or Flt3-expressing colonies, suggesting that Akt does not fully complement the proliferative function of IL-3 or of Flt3-ITD. Correct localization of Akt to the cell membrane was an absolute requirement, as the myristylation-defective (mislocalized) form of Akt was inactive (Fig. 2A and B and Fig. 3A-D). We previously showed that wtFlt3-expressing 32D cells did not form colonies after treatment with Flt3 ligand (14). As Akt activation was observed after Flt3 ligand stimulation (Fig. 1A), we speculate that sustained activation of Akt above a certain threshold is necessary for myeloid transformation, as provided by Flt3-ITD or by myristylated Akt.
The important and novel finding that activation of Akt was sufficient to mediate myeloid transformation in vitro was further underlined by our in vivo results (Fig. 4A; refs. 14, 27). In previous work, we showed that Flt3-ITD expression promoted a leukemia-like disease, whereas expression of wtFlt3 did not (14). As shown in Fig. 4A, activation of Akt was sufficient to cause a rapid onset of a leukemia-like disease and death, similarly to Flt3-ITD. Histologic examination revealed massive infiltration of the bone marrow with p-Akt-positive blasts (Fig. 4B), as well as of the liver and spleen. Onset of disease and death in activated Akt-ER* mice was observed earlier than in Flt3-ITD-bearing mice (Fig. 4A), which may be due to high Akt activation in vivo. Because Flt3-ITD mediate the activation of multiple aberrant signaling pathways, it would be interesting to compare the effects of Akt with Flt3-ITD in primary bone marrow.
Frequent activation of the PI3K/Akt pathway has been observed in lysates from AML patients (31) and found to predict poor overall survival (32). However, it remained unclear whether the blast population itself, or surrounding stroma contained the majority of p-Akt. Immunohistochemical stainings of our tissue array revealed that the blast population contained the majority of p-Akt, whereas normal bone marrow did not. Interestingly, p-Akt staining was nuclear in a large portion of the samples (Fig. 5B). The significance of this finding is unknown. Importantly, p-Akt was found in AML samples with wtFlt3, suggesting a more general role of Akt in leukemogenesis. Other signaling pathways such as the Ras pathway are likely to activate Akt in human AML. Oncogenic N-Ras and K-Ras mutations are found in a quarter of all cases of AML but rarely occur together with Flt3-ITD (9), suggesting functional overlap. As oncogenic Ras is known to activate the PI3K pathway (10), it is tempting to speculate that Akt may be a necessary point of convergence of oncogenic signaling required for leukemic transformation.
In conclusion, our data suggest that Akt activation and Foxo inactivation are important features of myeloid leukemogenesis. As Akt is necessary for transformation, the Akt kinase and Foxo transcription factors may be an attractive targets for therapeutic intervention.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank P. Coffer (Department of Immunology, University Medical Center Utrecht, Utrecht, the Netherlands) and D. Stokoe (Cancer Research Institute, University of California, San Francisco, CA) for plasmids; A. Finch, L. Swigart, and G. Evan for advice with the animal experiments; N. Adam for technical assistance; and D. Stokoe, A. Mirza, A. Becker, and S. Diederichs for discussion.
Received 2/ 8/05. Revised 7/15/05. Accepted 8/25/05.
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