The NPM/ALK fusion gene, formed by the t(2;5) translocation in a subset of anaplastic large cell lymphomas, encodes a Mr 75,000 hybrid protein that contains the NH2-terminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocations. Our studies showed that NPM/ALK, similar to other members of this family, activates phosphatidylinositol 3-kinase (PI3K) and its downstream effector, serine/threonine kinase (Akt). PI3K was found in complex with NPM/ALK. Both PI3K and Akt kinase were permanently activated in NPM/ALK-transfected BaF3 murine hematopoietic cells and in NPM/ALK-positive, but not in NPM/ALK-negative, patient-derived anaplastic large cell lymphoma cell lines. In addition, Akt was phosphorylated/activated in protein samples isolated from four patients diagnosed with ALK-positive, T/null-cell lymphomas. The PI3K inhibitors wortmannin and LY294002 induced apoptosis in NPM/ALK+ cells but exerted only minor effects on the control BaF3 parental cells and peripheral blood mononuclear cells stimulated by growth factors. Furthermore, retroviral infection of NPM/ALK+ BaF3 cells with a dominant-negative PI3K mutant (Δp85) or a dominant-negative Akt mutant (K179M) inhibited proliferation and clonogenic properties of the infected cells. Finally, the Akt mutant (K179M) suppressed the tumorigenicity of NPM/ALK-transfected BaF3 cells injected into syngeneic mice. In conclusion, our data indicate that NPM/ALK constitutively activates the PI3K-Akt pathway and that this pathway plays an important role in the NPM/ALK-mediated malignant transformation.

Tyrosine kinases play an essential role in cell proliferation, apoptosis, differentiation, and malignant transformation (1). Their enzymatic activity, which stimulates numerous signaling pathways, is tightly regulated by various components, such as growth factors and/or stress factors, that are responsible for turning on/off the kinase function. Constitutive activation of these kinases can lead to prolonged stimulation of the signaling pathways, resulting in neoplastic transformation.

Chromosomal translocations are responsible for the expression of abnormal fusion proteins that possess constitutive tyrosine kinase activity, i.e., OTKs.3 These translocations occur often in hematopoietic cells, leading to their transformation and the development of leukemias or lymphomas (2). The best characterized example of an OTK is the BCR/ABL fusion protein, a product of the t(9;22) chromosomal translocation (Philadelphia chromosome), which is responsible for the induction of chronic myelogenous leukemia and a subset of acute lymphocytic and myelogenous leukemias (3, 4). Other members of the BCR/ABL-related OTK family are TEL/ABL (5), TEL/JAK2 (6), TEL/PDGFR (7), and NPM/ALK (8). All of these OTKs have structural similarities, which include an NH2-terminal domain responsible for constitutive oligomerization and activation of the associated tyrosine kinase of the COOH-terminal fusion partner. BCR/ABL, TEL/ABL, TEL/PDGFR, and TEL/JAK2 induce acute and chronic leukemias, usually of myeloid lineage (2, 3, 4, 5, 6, 7). In contrast, the NPM/ALK protein produced by the t(2;5) translocation is found in approximately two-thirds of the Ki(CD30)-positive ALCLs (8). NPM is a ubiquitously expressed protein responsible for protein shuttling between the cytoplasm and the nucleus (9). ALK is a receptor tyrosine kinase physiologically expressed by neural tissues (10, 11). NPM/ALK encodes a Mr 75,000 fusion protein that contains the NH2-terminal 117 amino acid residues of NPM joined to the entire cytoplasmic portion of ALK (8). The NPM/ALK fusion protein possesses constitutive tyrosine kinase activity and is able to transform murine hematopoietic cells and to induce lymphoma (12).

The mechanisms of NPM/ALK-mediated transformation are not well understood. Although NPM/ALK can associate with and phosphorylate several adaptor proteins such as SHC, Grb2, and IRS-1 (13, 14), their role (if any) in NPM/ALK-induced transformation is uncertain. Recently, Bai et al.(15) demonstrated that NPM/ALK constitutively activates phospholipase Cγ to induce cell proliferation. In addition to stimulating proliferation, NPM/ALK protected the IL-3-dependent hematopoietic cell line BaF3 from apoptosis in growth factor-free conditions, a necessary prerequisite for malignant transformation. Because it has been demonstrated previously that antiapoptotic pathways in hematopoietic cells transformed by the BCR/ABL OTK require activation of PI3K-Akt serine/threonine kinase (16, 17), we thought that investigation of the potential role of the PI3K-Akt pathway in NPM/ALK-mediated lymphomagenesis was warranted.

PI3K was discovered as an activity that phosphorylates phosphoinositols at the D-3′ position of the inositol ring and produces novel phosphoinositides (PI3P; Ref. 18). Purified PI3K was shown to be a heterodimer consisting of a Mr 85,000 (p85) regulatory subunit and a Mr 110,000 (p110) catalytic subunit (19). The mechanism of PI3K activation is not fully understood, but association of p85 with various activated tyrosine kinases is thought to provide a signal sufficient for the activation of the p110 catalytic subunit. The PI3P generated by the activated p110 subunit is able to bind to the pleckstrin homology domains of several downstream signaling molecules including Akt (20), which is a major downstream effector of PI3K. Activation of Akt by PI3K involves binding of PI3Ps to the pleckstrin homology domain of Akt, which results in the translocation of Akt to the plasma membrane (21). Activation also includes phosphorylation of Akt on Thr-308 and Ser-473 by the PDK1 and PDK2 kinases (22, 23).

Here we show that the PI3K-Akt pathway is constitutively activated in NPM/ALK-transformed murine hematopoietic cell lines and in cell lines and lymphoma tissues from ALK-positive ALCL patients. In addition, we demonstrate that both PI3K and Akt are essential for the growth factor independence and lymphomagenic activity of NPM/ALK-transfected cells.

Plasmids.

Retroviral construct pSRαMSVtkneo-NPM/ALK containing the full-length NPM/ALK cDNA was described previously (12). The dominant-negative mutant of PI3K (Δp85; Ref. 24) was obtained from Dr. Michael D. Waterfield (Ludwig Institute for Cancer Research, London, United Kingdom) and cloned into the EcoRI site of the pSRαMSVtkneo plasmid. The dominant-negative mutant of Akt(K179M) linked to HA has been described (17, 25).

Cells.

The murine growth factor-dependent pro-B lymphoid cell line BaF3 was maintained in RPMI 1640 supplemented with 10% FBS and 15% WEHI-conditioned medium as a source of IL-3. BaF3 cell lines transfected with NPM/ALK (BaF3-NPM/ALK cells) or with empty virus (BaF3-neo) were obtained by electroporation of BaF3 parental cells with pSRαMSVtkneo-NPM/ALK or pSRαMSVtkneo plasmid, respectively, and selection in G418. Expression of NPM/ALK was confirmed by Western blot assay. BaF3 cells expressing TEL/JAK2 or TEL/ABL were obtained from Dr. Gary Gilliland (Harvard Medical School, Boston, MA), and BaF3-expressing TEL/PDGFR cells were obtained from Dr. Martin Carroll (University of Pennsylvania, Philadelphia, PA). BaF3 cell lines transfected with various OTKs were maintained in RPMI 1640 supplemented with 10% FBS and 15% WEHI-conditioned medium. ALCL cell lines carrying the t(2;5) chromosomal translocation and expressing NPM/ALK (Karpas 299, SUDHL-1, and JB6) and NPM/ALK-negative ALCL cell lines (PB-1, 2A, and 2B) were grown in RPMI 1640 supplemented with 10% FBS. The latter cell lines have been established from the patient at the early stage (PB-1) and at the late aggressive stage (2A and 2B) of T-cell lymphoma involving skin (26). PBMNCs were obtained after centrifugation on Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) and incubated in the presence of recombinant human IL-2 and IL-7 (Genetics Institute, Inc., Cambridge, MA). Lymph node biopsies were obtained from patients with ALCL and cryopreserved at −70°C. The diagnosis of ALCL was established on the basis of morphology and immunohistochemistry including positive staining with a monoclonal anti-ALK antibody (Dako, Carpinteria, CA) in all four cases.

Enzymatic Assays.

Cell lysates were obtained after 5 h starvation of the cells from growth factor and serum. For the PI3K assay, PI-3k was assayed in anti-phosphotyrosine immunoprecipitates using [γ-32P]ATP and phosphatidylinositol as a substrate as described (16). 32P-labeled PI3P was resolved by TLC and visualized by autoradiography. For the Akt assay, enzymatic activity of Akt was examined as described previously (17, 25). Briefly, anti-Akt immunoprecipitates were incubated with [γ-32P]ATP and histone H2B as a substrate. Reaction mixtures were electrophoresed in SDS-PAGE, transferred onto nitrocellulose membranes (Protran), and exposed to X-ray film. The amount of immunoprecipitated Akt was determined by Western blot.

Immunoprecipitation.

Cell lysates were prepared as described (16). The p85 subunit of PI3K and NPM/ALK were immunoprecipitated with rabbit anti-p85 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) or with goat anti-ALK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), respectively. Immunoprecipitates were analyzed by SDS-PAGE and Western blot assays using anti-ALK antibody and anti-p85 antibody, respectively. Immunoprecipitated p85 and NPM/ALK were detected by Western blot assay with anti-p85 or anti-ALK antibody, respectively.

Detection of Phosphorylated Akt.

Total cell lysates were obtained from lymphoma biopsies harvested from patients with ALCLs and analyzed by SDS-PAGE and Western blot assay using anti-phospho-Akt antibody, followed by anti-Akt antibody (both from New England Biolabs, Beverly, MA).

PI3K inhibitors.

WT and LY (both from Sigma) were dissolved in DMSO and added to the cultures (105 cells/ml/24-well plate) in the desired concentrations at 0, 12, and 24 h. DMSO was added to the control groups. The number of viable cells was evaluated at 48 h using trypan blue exclusion vital dye. The percentage of apoptotic cells was determined at the same time using the TACS1 Klenow in situ apoptosis detection kit (Trevigen), according to the manufacturer’s protocol. Cell cycle distribution was assayed after staining of DNA with propidium iodide as described (27).

Retroviral Infections.

Infections were performed as described previously (27) with some modifications. To increase the concentration of the virus, cells were suspended in medium collected from the culture of Bosc23 packaging cells transfected with the appropriate constructs and were cocultivated in the presence of IL-3 on a monolayer of freshly transfected Bosc23 cells. Bulk cultures obtained 72 h after infection were incubated for 24 h with 1 μg/ml of puromycin. After an additional 48 h, viable cells were isolated by centrifugation on Lympholyte M gradient medium (Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada) and used for experiments. Expression of Δp85 and Akt(K179M) in transfected cells was confirmed by Western blot assay detecting the presence of the truncated form of p85 or the Akt(K179M)-HA protein.

Clonogenic Assay.

Clonogenic assay was performed in MethoCult H4230 semisolid medium (Stem Cell Technologies, Vancouver, British Columbia, Canada) as described (17, 27) in the absence (BaF3-NPM/ALK cells) or in the presence (BaF3-neo cells) of IL-3 (10% of WEHI-conditioned medium). Colonies were counted after 7 days.

Tumorigenesis in Mice.

BALB/c female mice (Taconic Farms, Inc., Germantown, NY) were injected i.v. with 106 BaF3-NPM/ALK cells infected with LXSN-Akt(K179M) retrovirus or with LXSN retrovirus (15 mice/group). Terminally ill mice were sacrificed and examined for the development of hematological malignancy. Tissue sections from bone marrow, spleen, liver, lymph nodes, lungs, kidneys, pancreas, intestine, and brain were analyzed as described (17, 27, 28). In addition, selected slides were stained with anti-ALK antibody to confirm the tissue involvement by lymphoma.

NPM/ALK Activates PI3K and Akt.

After stimulation by tyrosine kinases, PI3K generates PI3Ps, which function as secondary messengers in signaling pathways (29). In the absence of growth factors, cell lines expressing NPM/ALK or other members of the BCR/ABL-related OTK family, but not parental BaF3 cells transfected with empty vector, contained an activated form of PI3K able to produce PI3P (Fig. 1, top panel). Because PI3K induces Akt kinase activity, we analyzed the activation status of Akt in these cells. In vitro kinase assay using [γ-32P]ATP and histone 2B as a substrate demonstrated that the enzymatic activity of Akt was increased in cells expressing NPM/ALK or the other members of the BCR/ABL-related OTK family when compared with the parental BaF3 cells (Fig. 1, bottom panel). Activation of PI3K by NPM/ALK appears to occur probably via direct interaction, because coimmunoprecipitation studies showed that the p85 subunit of PI3K is in a complex with NPM/ALK (Fig. 2 A).

PI3K and Akt were enzymatically activated not only in BaF3-NPM/ALK cells but also in NPM/ALK-positive cell lines (Karpas 299, SUDHL-1, and JB6) established from patients with ALCL containing the t(2;5) (Fig. 2,B), in comparison with BaF3 parental cells and PBMNCs. Because phosphorylation of Akt on Ser-473 is associated with its activation (22, 23), we used an anti-Akt phosphoserine-473 antibody to detect activated Akt. Phosphorylation of Akt on Ser-473 was found in all samples obtained from NPM/ALK-positive cells, including the lymph nodes infiltrated with lymphoma (Fig. 2 C). In contrast, Baf3 parental cells, PBMNCs and NPM/ALK-negative ALCL cell lines did not display phosphorylated Akt.

PI3K and Akt Are Involved in NPM/ALK-mediated Growth Factor Independence and Transformation.

To determine whether PI3K plays a role in NPM/ALK-induced growth factor independence, apoptosis and proliferation of NPM/ALK-transformed BaF3 cells and NPM/ALK-positive ALCL cell lines were assessed in growth factor-free conditions after exposure to the PI3K inhibitors WT or LY. WT induced massive apoptosis in NPM/ALK-transformed BaF3 cells grown in the absence of IL-3, about 90 or 100% of cells being apoptotic in 0.1 or 1 μm WT (Fig. 3, top left panel). In contrast, BaF3 parental cells cultured in the presence of IL-3 were much less sensitive, with only about 20 or 50% of these cells undergoing apoptosis in the same concentrations of WT. NPM/ALK-positive ALCL cells were also sensitive to WT; 75–80% of these cells died after exposure to a 1 μm concentration of the compound (Fig. 3, top right panel). Normal mononuclear leukocytes (PBMNCs), on the other hand, were relatively resistant to WT, with only 30% dying in the same concentration of WT. Although LY exerted similar effects as WT on BaF3-NPM/ALK cells (Fig. 3, bottom left panel), it induced less robust apoptosis in NPM/ALK-positive ALCL cell lines (Fig. 3, bottom right panel). Nonetheless, like WT, much greater apoptosis was observed in NPM/ALK-positive cells as compared with BaF3-neo or PBMNCs.

WT and LY also inhibited proliferation of NPM/ALK-positive cells (Fig. 4). This effect appeared to be attributable not only to the induction of apoptosis caused by these compounds because inhibition of the growth rate of NPM/ALK-transformed cells was usually much more profound than expected based on the results of the apoptosis assays (Fig. 3). For example, 0.1 μm WT or 20 μg/ml LY induced apoptosis in ∼30% of JB6 cells (Fig. 3, top right panel) and inhibited growth of these cells in about 80–90% (Fig. 4,A, top right panel), implicating the PI3K pathway in NPM/ALK-dependent proliferation. Cell cycle analysis confirmed this conclusion and revealed that PI3K inhibitors, in addition to apoptosis (sub-G1 peak), caused a reduction of the percentage of NPM/ALK-positive JB6 cells in S-phase and accumulation in G0-G1 phase (Fig. 4,B). Moreover, treatment with WT or LY inhibited colony formation ability of NPM/ALK-positive cells in semisolid medium and colonies that arose from treated cells were smaller as compared with those formed by untreated cells (data not shown). Low and moderate concentrations of both inhibitors exerted similar effects on the proliferation of BaF3-neo cells and BaF3-NPM/ALK cells, with the differential influence of WT and LY on the growth of these cells visible only at the highest concentrations tested (Fig. 4, left panels). However, growth factor-independent proliferation of NPM/ALK-positive ALCL cells was clearly more affected by WT and LY in all tested concentrations than the proliferation of PBMNCs in the presence of IL-2 and IL-7 (Fig. 4, right panels).

Because WT and LY can inhibit the enzymatic activity of other kinases such as ATM and DNA-PK (30) in addition to PI3K, dominant-negative mutants of PI-3k (Δp85) and Akt (K179M) were used to obtain direct evidence that the PI3K-Akt pathway is essential for NPM/ALK-mediated transformation. Infection of BaF3-NPM/ALK cells with retroviruses carrying Δp85 or Akt (K179M) dominant-negative mutants (Fig. 5,A) dramatically reduced their clonogenic capability in growth factor-free medium (Fig. 5,B). In comparison, expression of these mutants in BaF3-neo cells exerted only a moderate effect on their clonogenic potential in the presence of IL-3. The size of colonies, however, was reduced in the case of BaF3-NPM/ALK cells as well as BaF3-neo cells (Fig. 5 C).

PI3K-Akt Pathway Is Required for Tumorigenic Activity of NPM/ALK.

To determine the role of the PI3K-Akt pathway in NPM/ALK-mediated tumorigenesis, BaF3-NPM/ALK cells infected with retrovirus carrying the Akt (K179M) dominant-negative mutant or with empty virus were inoculated into syngeneic BALB/c mice. All mice receiving injections of BaF3-NPM/ALK + empty virus cells were sacrificed or died after 5–8 weeks because of the development of hematological malignancies (Fig. 6,A). By contrast, inoculation of BaF3-NPM/ALK + Akt (K179M) cells induced fatal disease in only one mouse; the other mice were alive and well after 30 weeks from the time of inoculation. Histopathological examination of the internal organs of the moribund mice revealed the presence of macroscopic and microscopic tumor foci in liver, spleen, brain, and meninges. Histologically, the tumors were comprised of sheets of lymphoid cells with large nuclei, prominent nucleoli, and a moderate amount of cytoplasm (Fig. 6 B). Immunohistochemical staining confirmed the presence in tumor cells of highly expressed ALK protein, representing the product of the transfected NPM/ALK gene. No tumors were seen either macroscopically or microscopically in the organs derived from the healthy appearing mice that received injections of NPM/ALK-positive cells also expressing the Akt (K179M) dominant-negative mutant.

NPM/ALK is an oncogenic tyrosine kinase resulting from the t(2;5) chromosomal translocation associated with ALCL (8). The transforming capacity of NPM/ALK was confirmed by the studies of Kuefer et al.(12), demonstrating that NPM/ALK induces lymphoma when expressed in murine hematopoietic cells. However, the mechanisms of NPM/ALK-mediated malignant transformation are largely unknown.

Recently, adaptor proteins, such as Grb-2, IRS-1, and Shc (12, 13), and the signal transduction protein phospholipase Cγ (14), have been implicated in NPM/ALK-induced lymphomagenesis. These proteins are also essential for the regulation of growth and differentiation of normal hematopoietic cells, as well as cells transformed by BCR/ABL (31). In previous studies, we were able to demonstrate that other signaling molecules, PI3K and its downstream effector Akt, are also essential for leukemogenesis mediated by the BCR/ABL oncogenic tyrosine kinase (16, 17). Surprisingly, PI3K was found to be expendable in normal hematopoietic cells (16), suggesting that this kinase could be a potential target for novel antileukemia treatment.

In this report, we show that PI3K and Akt are activated in the murine lymphocytic cell line BaF3 transformed by NPM/ALK and also by other members of the BCR/ABL-related OTK family including BCR/ABL, TEL/ABL, TEL/JAK2, and TEL/PDGFR. This finding suggests that activation of the PI3K-Akt pathway may be a common phenomenon occurring in hematopoietic cells transformed by OTKs. In addition, we demonstrate that PI3K and/or Akt are stimulated in ALCL cell lines bearing the t(2;5) chromosomal translocation (Karpas 299, SUDHL-1, and JB6) and, most importantly, also in fresh specimens of lymph nodes containing ALK+ ALCL cells. Interestingly, Akt was not activated in NPM/ALK-negative ALCL cells (PB-1, 2A, and 2B), which suggests that the PI3K-Akt pathway is selectively activated in ALCLs bearing t(2;5) translocation. Activation of the PI3K/Akt pathway in NPM/ALK-transformed cells is probably initiated by interaction of NPM/ALK with the p85 subunit of PI3K (this report), because association of p85 with a tyrosine kinase invariably triggers activation of the p110 catalytic subunit of PI3K (29).

To study the importance of the PI3K-Akt pathway for the transforming capacity of NPM/ALK, we used two selective inhibitors of PI3K activity, WT and LY (32, 33). Both compounds were able to induce apoptosis in NPM/ALK-transformed BaF3 and NPM/ALK-positive ALCL cell lines, demonstrating that PI3K activity is essential for the antiapoptotic properties of NPM/ALK. However, NPM/ALK-positive ALCL cell lines were generally less sensitive to apoptosis induced by low and moderate doses of WT or LY in comparison with BaF3-NPM/ALK cells. In addition to the stimulation of apoptosis, WT and LY blocked NPM/ALK-induced growth factor-independent proliferation, and again NPM/ALK-positive ALCL cells appeared to be less sensitive than BaF3-NPM/ALK cells to these inhibitors. The lower susceptibility of the former cells to the growth-inhibitory and proapoptotic effects of WT and LY might potentially be explained by differences in processing of these inhibitors in the cells (uptake, distribution, and degradation). However, redundancy in signaling pathways regulating apoptosis in NPM/ALK-positive ALCL cells cannot be ruled out, because although no significant apoptosis was observed in these cells in the presence of 0.1 μm WT or in 20 μg/ml LY, their growth ability was severely affected.

Interestingly, PBMNCs stimulated with IL-2 and IL-7 were not significantly susceptible to concentrations of WT or LY that strongly affected the spontaneous growth of NPM/ALK-positive ALCL cells. IL-3-stimulated BaF3 cells were also less sensitive to a high concentration of WT and LY than BaF3-NPM/ALK cells growing in the absence of IL-3. However, growth of these cells was equally affected by low and moderate concentrations of the drugs. These results are in agreement with our original observation that WT does not affect the survival of normal bone marrow cells stimulated by various cytokines, although inducing apoptosis in BCR/ABL-transformed cells (16). In addition, although growth factor-dependent proliferation of primary hematopoietic cells was not affected by WT, proliferation of growth factor (IL-3, granulocyte/macrophage-colony stimulating factor, and stem cell factor)-dependent cell lines was inhibited by WT. In agreement, Santos et al.(34) showed recently that LY does not induce apoptosis in BaF3 cells cultured in the presence of IL-3 but does inhibit proliferation of these cells. Overall, these observations suggest that inhibitors of PI3K might be useful in the treatment of leukemias or lymphomas caused by BCR/ABL or NPM/ALK and perhaps also of malignancies induced by other members of the BCR/ABL-related OTK family.

To provide more direct evidence for the role of the PI3K-Akt pathway in NPM/ALK-mediated transformation and to exclude the involvement of other WT- and LY-sensitive kinases such as ATM and DNA-PK (30), dominant-negative mutants of PI3K and Akt were used in our assays. These studies clearly demonstrated that PI3K-Akt signaling is essential for NPM/ALK-mediated transformation. In addition, inhibition of Akt almost completely abrogated the tumorigenic capability of NPM/ALK-transformed cells in syngeneic mice. In agreement with Craddock et al.(35), we show that IL-3-dependent clonogenic capability of nontransformed BaF3-neo cells was only moderately inhibited by the PI3K and Akt dominant-negative mutants, although the size of colonies was reduced significantly.

In conclusion, we have demonstrated that NPM/ALK, similar to the other members of the BCR/ABL-related OTK family, activates the PI3K-Akt pathway, which appears to be essential for its transforming ability. Given that treatment of normal hematopoietic cells with PI3K inhibitors does not induce significant effects in such cells, although inhibiting growth and inducing apoptosis in cells transformed by BCR/ABL (16), by NPM/ALK (this report), and by other members of the BCR/ABL-related OTK family, such as TEL/ABL, TEL/JAK2 and TEL/PDGFR (our unpublished observation),4 these findings support the hypothesis that the PI3K-Akt pathway, and specifically PI3K, could be an attractive target for novel antitumor therapies.

Fig. 1.

NPM/ALK and other OTKs activate PI3K and Akt. BaF3-neo cells (Neo) and BaF3 cells expressing various OTKs were starved from growth factors and serum for 5 h. PI3K (top panel) and Akt (bottom panel) activity was then measured by in vitro kinase assays using phosphatidylinositol and histone 2B (H2B) as substrates, respectively. Immunoprecipitated Akt was detected by Western blot assay. Results are representative of two experiments.

Fig. 1.

NPM/ALK and other OTKs activate PI3K and Akt. BaF3-neo cells (Neo) and BaF3 cells expressing various OTKs were starved from growth factors and serum for 5 h. PI3K (top panel) and Akt (bottom panel) activity was then measured by in vitro kinase assays using phosphatidylinositol and histone 2B (H2B) as substrates, respectively. Immunoprecipitated Akt was detected by Western blot assay. Results are representative of two experiments.

Close modal
Fig. 2.

NPM/ALK associates with PI3K and activates the PI3K-Akt pathway in ALCL cells. A, p85 subunit of PI3K was immunoprecipitated from BaF3-neo cells (Lane 1), BaF3-NPM/ALK cells (Lane 2), and Karpas 299 cells (Lane 4). The presence of NPM/ALK was detected by Western blot assay with anti-ALK antibody (top panels). NPM/ALK was not visible in the control nonimmune precipitates (Lanes 3 and 5). NPM/ALK was immunoprecipitated from Karpas 299 cells (Lane 6), and p85 was detected by Western blot assay (top panel). p85 was not present in the control nonimmune precipitate (Lane 7). The presence of p85 and NPM/ALK in the specific immunoprecipitates was detected by Western blot assay (bottom panels). B, PI3K (top panel) and Akt (bottom panel) enzymatic activities were measured in BaF3-NPM/ALK cells and also in human cell lines established from NPM/ALK-positive ALCLs, as described in the legend to Fig. 1. BaF3-neo cells (Neo) and PBMNCs were used as negative controls. H2B, histone 2B. C, phosphorylated (activated) Akt was detected by Western blot assay using antiphospho-Akt antibody with lysates isolated from BaF3-NPM/ALK cells, NPM/ALK-positive ALCL cell lines, and lymph node specimens containing ALK+ ALCLs but not in BaF3-neo parental cells, PBMNCs, and NPM/ALK-negative ALCL cells. Results are representative of three separate experiments.

Fig. 2.

NPM/ALK associates with PI3K and activates the PI3K-Akt pathway in ALCL cells. A, p85 subunit of PI3K was immunoprecipitated from BaF3-neo cells (Lane 1), BaF3-NPM/ALK cells (Lane 2), and Karpas 299 cells (Lane 4). The presence of NPM/ALK was detected by Western blot assay with anti-ALK antibody (top panels). NPM/ALK was not visible in the control nonimmune precipitates (Lanes 3 and 5). NPM/ALK was immunoprecipitated from Karpas 299 cells (Lane 6), and p85 was detected by Western blot assay (top panel). p85 was not present in the control nonimmune precipitate (Lane 7). The presence of p85 and NPM/ALK in the specific immunoprecipitates was detected by Western blot assay (bottom panels). B, PI3K (top panel) and Akt (bottom panel) enzymatic activities were measured in BaF3-NPM/ALK cells and also in human cell lines established from NPM/ALK-positive ALCLs, as described in the legend to Fig. 1. BaF3-neo cells (Neo) and PBMNCs were used as negative controls. H2B, histone 2B. C, phosphorylated (activated) Akt was detected by Western blot assay using antiphospho-Akt antibody with lysates isolated from BaF3-NPM/ALK cells, NPM/ALK-positive ALCL cell lines, and lymph node specimens containing ALK+ ALCLs but not in BaF3-neo parental cells, PBMNCs, and NPM/ALK-negative ALCL cells. Results are representative of three separate experiments.

Close modal
Fig. 3.

PI3K inhibitors induce apoptosis in NPM/ALK-transformed cells. WT and LY were added to BaF3-NPM/ALK cells growing in the absence of IL-3 to NPM/ALK-positive ALCL cell lines cultured in 10% FBS-containing medium and to BaF3-neo cells (Neo) and PBMNCs cultured in the presence of IL-3 or IL-2 + IL-7, respectively. Apoptotic cells were detected using an in vitro apoptosis detection kit. Results represent three separate experiments.

Fig. 3.

PI3K inhibitors induce apoptosis in NPM/ALK-transformed cells. WT and LY were added to BaF3-NPM/ALK cells growing in the absence of IL-3 to NPM/ALK-positive ALCL cell lines cultured in 10% FBS-containing medium and to BaF3-neo cells (Neo) and PBMNCs cultured in the presence of IL-3 or IL-2 + IL-7, respectively. Apoptotic cells were detected using an in vitro apoptosis detection kit. Results represent three separate experiments.

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Fig. 4.

PI3K inhibitors reduce the growth rate of NPM/ALK-transformed cells. Cells were treated as described in the Fig. 3 legend. A, viable cells were counted using trypan blue exclusion. Results represent three experiments and show the growth inhibition rate calculated as follows: (number of viable cells in the inhibitor-treated sample/number of viable cells in untreated sample) × 100%. B, cell cycle distribution of JB6 cells untreated (top panel) and treated with 0.1 μm WT (middle panel) or 20 μg/ml LY (bottom panel). Results represent two separate experiments.

Fig. 4.

PI3K inhibitors reduce the growth rate of NPM/ALK-transformed cells. Cells were treated as described in the Fig. 3 legend. A, viable cells were counted using trypan blue exclusion. Results represent three experiments and show the growth inhibition rate calculated as follows: (number of viable cells in the inhibitor-treated sample/number of viable cells in untreated sample) × 100%. B, cell cycle distribution of JB6 cells untreated (top panel) and treated with 0.1 μm WT (middle panel) or 20 μg/ml LY (bottom panel). Results represent two separate experiments.

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Fig. 5.

The PI3K-Akt pathway is essential for NPM/ALK-induced growth factor-independent proliferation. BaF3-neo cells (Neo) and BaF3-NPM/ALK cells (NPM/ALK) were infected with retroviruses carrying the Δp85 mutant (Lanes 3 and 4), Akt (K179M) mutant (Lanes 7 and 8), or with empty virus (Control; Lanes 1, 2, 5, and 6). A, expression of Δp85 mutant and Akt (K179M)-HA mutant was detected by Western blot assay with anti-p85 antibody (top panel) and anti-Akt antibody (bottom panel). The expression of Akt (K179M)-HA was confirmed by Western blot with anti-HA antibody (data not shown). B, infected cells were plated in methylcellulose, and colonies were counted after 7 days. Results represent the number of colonies from three separate experiments; bars, SD. C, pictures of the representative single colonies.

Fig. 5.

The PI3K-Akt pathway is essential for NPM/ALK-induced growth factor-independent proliferation. BaF3-neo cells (Neo) and BaF3-NPM/ALK cells (NPM/ALK) were infected with retroviruses carrying the Δp85 mutant (Lanes 3 and 4), Akt (K179M) mutant (Lanes 7 and 8), or with empty virus (Control; Lanes 1, 2, 5, and 6). A, expression of Δp85 mutant and Akt (K179M)-HA mutant was detected by Western blot assay with anti-p85 antibody (top panel) and anti-Akt antibody (bottom panel). The expression of Akt (K179M)-HA was confirmed by Western blot with anti-HA antibody (data not shown). B, infected cells were plated in methylcellulose, and colonies were counted after 7 days. Results represent the number of colonies from three separate experiments; bars, SD. C, pictures of the representative single colonies.

Close modal
Fig. 6.

Akt is essential for the development of NPM/ALK-mediated hematological malignancies. BaF3-NPM/ALK cells infected with Akt (K179M) virus or with empty virus (E) were injected i.v. into syngeneic BALB/c mice. A, survival time of the animals was monitored weekly. B, a representative tissue section from the liver of a mouse injected with BaF3-NPM/ALK cells was stained with H&E (top left and right panels, low and high magnifications, respectively). NPM/ALK+ tumor cells were detected by immunohistochemistry using anti-ALK antibody and show brown staining (bottom left and right panels, low and high magnifications, respectively).

Fig. 6.

Akt is essential for the development of NPM/ALK-mediated hematological malignancies. BaF3-NPM/ALK cells infected with Akt (K179M) virus or with empty virus (E) were injected i.v. into syngeneic BALB/c mice. A, survival time of the animals was monitored weekly. B, a representative tissue section from the liver of a mouse injected with BaF3-NPM/ALK cells was stained with H&E (top left and right panels, low and high magnifications, respectively). NPM/ALK+ tumor cells were detected by immunohistochemistry using anti-ALK antibody and show brown staining (bottom left and right panels, low and high magnifications, respectively).

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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.

1

Supported in part by NIH Grant CA70815, ACS RPG 98-348-01, and Grant 501-1-1-03-07/00 from the Medical Center for Postgraduate Education (all to T. S.); by NIH Grant CA089194 (to M. A. W.); by NIH Grant CA69129 and CORE Grant CA21765 (to S. W. M.); and by the American-Lebanese Syrian Associated Charities, St. Jude Children’s Research Hospital. T. S. is the Leukemia and Lymphoma Society Scholar, A. S. is a recipient of the fellowship from the Leukemia Research Foundation, and M. A. W. is a recipient of a Shannon Award from the National Cancer Institute.

3

The abbreviations used are: OTK, oncogenic tyrosine kinase; PDGFR, platelet-derived growth factor receptor; NPM, nucleolar phosphoprotein nucleophosmin; ALK, anaplastic lymphoma kinase; ALCL, anaplastic large cell lymphoma; IL, interleukin; PI3K, phosphatidylinositol 3-kinase; PI3P, phosphatidylinositol phosphate; HA, hemagglutinin antigen; PBMNC, peripheral blood mononuclear cell; WT, wortmannin; LY, LY294002.

4

A. Slupianek and T. Skorski, unpublished observation.

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