Activation of Mammalian Target of Rapamycin Signaling Pathway Contributes to Tumor Cell Survival in Anaplastic Lymphoma Kinase–Positive Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) is an aggressive form of malignant lymphoma of T/null lineage (1). A subset of ALCL tumors carries chromosomal aberrations involving the anaplastic lymphoma kinase (alk) gene on chromosome 2p23 resulting in ALK overexpression (2). The most common of these aberrations is the t(2;5)(p23;q35), which results in expression of the chimeric protein, nucleophosmin-ALK (3). Recent studies have established that nucleophosmin-ALK mediates oncogenesis, at least in part, through phosphorylation/activation of the serine/threonine kinase AKT (4, 5). AKT is a downstream effector of phosphatidylinositol 3-kinase (PI3K) and a critical mediator of mammalian target of rapamycin (mTOR) activity (6). However, the activation status of the mTOR pathway in ALK+ ALCL is unknown. Studying the status of the mTOR pathway in ALK+ ALCL is important because it may provide a rationale for a novel therapeutic strategy for these tumors.

mTOR is a serine/threonine protein kinase that forms two distinct protein complexes within cells: one with the regulatory-associated protein of TOR (raptor) and another with the rapamycin-insensitive companion (rictor; refs. 7, 8). mTOR-raptor complex (composed of mTOR, raptor, and GβL) is sensitive to rapamycin and regulates cell growth, in part, by phosphorylation of the ribosomal protein S6 (rpS6) kinase (p70S6K or S6K1) and subsequent phosphorylation of rpS6 to stimulate protein translation and ribosome biogenesis (9, 10). mTOR-raptor also leads to phosphorylation and inactivation of the eukaryotic initiation factor 4E (eiF4E)–binding protein-1 (4E-BP1), dissociating 4E-BP1 from the RNA cap-binding protein eIF4E, thus promoting cap-dependent translation of mRNA (7, 11, 12). mTOR-rictor complex (composed of mTOR, rictor, and GβL) is not rapamycin sensitive and modulates cell survival and proliferation by direct phosphorylation of AKT on Ser⁴⁷³ and by facilitating AKT phosphorylation on Thr³⁰⁸ by PDK1 in vitro (13).

Rapamycin is a macrolide antibiotic with antitumor activity (14, 15). Recent in vitro and in vivo studies have shown that rapamycin and its analogues have substantial antitumor activity in hematologic malignancies (16, 17). The mechanism by which rapamycin inhibits mTOR-raptor complex kinase activity is not completely understood. Rapamycin forms an inhibitory complex with the FKBP-rapamycin-binding domain of mTOR, which probably destabilizes the mTOR-raptor interaction and prevents mTOR from phosphorylating p70S6K and 4E-BP1 (18, 19).

In this study, we show that the mTOR pathway is frequently activated in ALK+ ALCL cell lines and tumors and that PI3K/AKT contributes to activation of the mTOR pathway in vitro. We also show that inhibition of mTOR expression or activity markedly decreased activation of the mTOR pathway associated with cell cycle arrest and apoptosis in ALK+ ALCL cells. These findings suggest that activation of mTOR signaling contributes to tumor cell survival in ALK+ ALCL, thus providing a potential therapeutic target in this lymphoma type.

Cell lines and reagents. Two ALK+ ALCL cell lines were used, Karpas 299 and SU-DHL1. The cell lines were maintained at 37°C in RPMI 1640 supplemented with 10% FCS in a humidified atmosphere containing 5% CO₂. The PI3K inhibitor, LY294002, the selective AKT inhibitor AKT II, and the mTOR-raptor inhibitor, rapamycin, were purchased from Calbiochem (San Diego, CA). Karpas 299 and SU-DHL1 cells were treated with LY294002 at a concentration of 0, 10, or 20 μg/μL as described elsewhere (5), rapamycin at a concentration of 0, 10, 20, and 40 μg/μL with or without 1 μg/μL doxorubicin, and AKT-II inhibitor (Calbiochem; ref. 20) as indicated. Whole-cell lysates were prepared 24 and 48 hours following treatment.

Immunohistochemistry. Rabbit polyclonal antibodies to total AKT (Santa Cruz Biotechnology, Santa Cruz, CA); mTOR, Ser²⁴⁴⁸p-mTOR, Ser²⁴⁰/Ser²⁴⁴p-rpS6, Thr⁷⁰p-4E-BP1, total 4E-BP1, and total eIF4E (Cell Signaling Technology, Beverly, MA); and mouse monoclonal antibodies to Ser⁴⁷³p-AKT and Thr³⁸⁹p-p70S6K (Cell Signaling Technology) were used. ALK expression was assessed using the mouse monoclonal antibody ALK-1 (DakoCytomation, Carpinteria, CA). Immunohistochemical methods were used to assess protein expression in formalin-fixed, paraffin-embedded cell blocks from Karpas 299 and SU-DHL1 and in 31 ALK+ ALCL tumors using methods described previously (21). The diagnosis of ALCL was based on the criteria of the WHO classification (1). Each neoplasm was part of a tissue microarray constructed as described elsewhere (22).

Western blot analysis. Subcellular fractionation was done using the NE-PER nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL) according to the manufacturer's protocol with minor modifications. Western blot analysis was done using standard methods as described previously (21). Rabbit polyclonal antibodies to total AKT (Santa Cruz Biotechnology), rabbit polyclonal antibodies to Ser²⁴⁴⁸p-mTOR, p70S6K, Ser²⁴⁰/Ser²⁴⁴p-rpS6, Thr⁷⁰p-4E-BP1, total 4E-BP1, and total eIF4E (Cell Signaling Technology), mouse monoclonal antibodies to Ser⁴⁷³p-AKT and Thr³⁸⁹p-p70S6K (Cell Signaling Technology), and rabbit monoclonal antibodies to rpS6 (Cell Signaling Technology) were used. Mouse monoclonal antibodies were used for p21, MCL-1, BCL-2 (DakoCytomation), p27, cyclin A (BD Biosciences PharMingen, San Diego, CA), BCL-XL (Zymed, South San Francisco, CA), c-FLIP, hemagglutinin (HA; Santa Cruz Biotechnology), and β-actin (Sigma, St. Louis, MO).

Adenovirus infection. Karpas 299 and SU-DHL1 cells were infected with the HA-tagged, constitutively active, adeno-myrAkt adenovirus (23) at a multiplicity of infection (MOI) of 20, shown previously to significantly increase Ser⁴⁷³p-AKT levels in our in vitro system (data not shown). Whole-cell lysates were prepared from control and infected cells 48 hours after infection. Expression of adeno-myrAkt in infected cells was confirmed by Western blot analysis using the anti-HA antibody. Infection of Karpas 299 and SU-DHL1 cells with a recombinant adenovirus construct expressing β-Gal (adeno-β-Gal) at the same MOI was used as an additional control in these experiments. Adeno-β-Gal was produced using the Adeno-X TRE-β-Gal virus stock (Clontech, Palo Alto, CA).

Inhibition of mTOR and AKT1 expression with small interfering RNA. The sequences of small interfering RNA (siRNA) targeting the human mTOR, AKT1, and 4E-BP1 gene products were purchased from Ambion, Inc. (Austin, TX) and were as follows: mTOR sense GGAGUCUACUCGCUUCUAUTT and antisense AUAGAAGCGAGUAGACUCCTC, AKT1 sense GGGCACUUUCGGCAAGGUGTT and antisense CACCUUGCCGAAAGUGCCCTT, and 4E-BP1 sense GGUACCAGGAUCAUCUAUGTT and antisense CAUAGAUGAUCCUGGUACCTC. The negative control 2 siRNA (Ambion) was also used.

Transient transfections of Karpas 299 and SU-DHL1 cells were done using the Nucleofector solution “T” protocol and appropriate program recommended by Amaxa Biosystems (Gaithersburg, MD) and concentrations of siRNAs as indicated. Cells were harvested at 48 hours following transient transfections with the siRNAs and whole-cell lysates were prepared. Western blot analysis confirmed adequate inhibition of protein expression in transiently transfected cells.

Cell viability and apoptosis studies. Cell viability was evaluated using trypan blue exclusion assay in triplicate. Annexin V staining (BD Biosciences PharMingen) detected by flow cytometry was used to assess apoptosis according to the manufacturer's instructions. Briefly, the cells were washed in ice-cold PBS and resuspended in binding buffer at a concentration of 1 × 10⁶ cells/mL. Subsequently, aliquots of 100 μL (1 × 10⁵ cells/mL) were incubated with 5 μL Annexin V-FITC and 5 μL propidium iodide for 15 minutes in the dark at room temperature and 1 × 10⁴ ungated cells were counted using a flow cytometer (FACSCalibur, Becton Dickinson). All experiments were done in triplicate.

Proliferation assay. Karpas 299 and SU-DHL1 cells were treated with LY294002 or rapamycin in 12-well plates using different concentrations as indicated. At 48 hours, a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), was added to each well and MTS-positive cells were counted using the CellTiter 96 AQ_ueous cell proliferation assay (Promega, Madison, WI) and μQuant spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT) according to the manufacturer's instructions.

Bromodeoxyuridine incorporation assay. Cell cycle S-phase fraction was assessed by a colorimetric bromodeoxyuridine incorporation assay. Briefly, 10⁴ cells per well were incubated with bromodeoxyuridine diluted 1:100 in 96-well plates for 1 to 1.5 hours at 37°C. An anti-bromodeoxyuridine antibody peroxidase conjugate (Roche Molecular Biochemicals, Mannheim, Germany) was used at a 1:200 dilution according to the manufacturer's recommended protocol. After appropriate washings, the colorimetric reaction was achieved using a substrate (tetramethylbenzidine) and evaluated using a plate reader (μQuant spectrophotometer, Bio-Tek Instruments). Multiple readings were obtained every 5 minutes for 30 minutes to ensure that the colorimetric reaction had reached its end point.

Statistical analysis. χ² and Fisher's exact tests were used to compare the expression among the mTOR signaling proteins in ALK+ ALCL tumors. Statistical calculations were done using StatView (Abacus Concepts, Inc., Berkeley, CA).

mTOR pathway is frequently activated in ALK+ ALCL tumors and cell lines. The phosphorylation status of mTOR, 4E-BP1, p70S6K, and rpS6 was immunohistochemically examined in 31 ALK+ ALCL tumors. We also determined the expression of total 4E-BP1 and eIF4E in these tumors. We found that mTOR signaling is frequently activated in ALK+ ALCL (Fig. 1). p-mTOR was detected in 24 of 29 (82.8%) and p-p70S6K (cytoplasmic or nuclear) in 24 of 30 (80%) cases (Table 1). The phosphorylation levels of p-mTOR and p-p70S6K as detected by immunohistochemistry were significantly associated (P = 0.0026, χ² test), consistent with previous studies on the causal association of these molecules (24). p-rpS6 was detected in 24 of 31 (77.4%) cases and its phosphorylation level was significantly associated with the phosphorylation level of p-4E-BP1 and total eIF4E (P = 0.016 and 0.029, respectively, χ² test). Codetection of all four phosphorylated proteins was seen in 19 of 29 cases (66%; Table 1). Of note, AKT activation in ALK+ ALCL tumors, determined as Ser⁴⁷³p-AKT expression (20), significantly correlated with p-mTOR and p-rpS6 (P = 0.037 and 0.002, respectively, χ² test) as well as with total 4E-BP1 and total eIF4E (P = 0.001 and 0.003, respectively, χ² test). p-4E-BP1 and total eIF4E were both positive in all 31 ALK+ ALCL tumors examined, but the level of detection was variable. eIF4E was strongly positive in 22 of 31 (71%) tumors.

We also assessed the baseline activation status of the mTOR pathway in Karpas 299 and SU-DHL1 cell lines. Western blot analysis revealed high levels of p-mTOR associated with high levels of p-p70S6K, p-rpS6, p-4E-BP1, and total eIF4E in both cell lines (Fig. 1).

Inhibition of PI3K/AKT down-regulates mTOR pathway. We assessed the ability of PI3K/AKT to contribute to mTOR pathway activation in Karpas 299 and SU-DHL1 cell lines. Treatment of these cell lines with the PI3K inhibitor, LY294002, resulted in a concentration-dependent decrease of the phosphorylation levels of AKT, mTOR, 4E-BP1, p70S6K, and S6K (Fig. 2A and B). These results show that mTOR signaling is regulated, at least in part, by PI3K/AKT and that inhibition of PI3K/AKT decreases the level of activation of mTOR signaling proteins.

Constitutively active AKT results in activation of mTOR pathway. To investigate if AKT activation is sufficient to activate the mTOR pathway in ALK+ ALCL cells, we infected the Karpas 299 and SU-DHL1 cell lines with an adenoviral vector, adeno-myrAkt, expressing constitutively active AKT (23). Constitutive activation of AKT resulted in a substantial increase in the phosphorylation of mTOR, p70S6K, and rpS6 (Fig. 3A and B).

Inversely, almost complete inhibition of AKT1 gene expression by AKT1-specific siRNA resulted in decreased p-mTOR levels (Fig. 3C). Similarly, inhibition of AKT activity using a specific AKT-II inhibitor (20) resulted in decreased p-mTOR levels (Fig. 3D). These results indicate that AKT activation contributes to activation of the mTOR pathway in this in vitro system.

Inhibition of mTOR-raptor complex by rapamycin and silencing mTOR by siRNA down-regulates mTOR signaling. Treatment of the Karpas 299 and SU-DHL1 cell lines with increasing concentrations of rapamycin, an inhibitor of the mTOR-raptor complex, resulted in a marked concentration-dependent decrease of the phosphorylation levels of mTOR, p70S6K, 4E-BP1, and total eIF4E (Fig. 4A). Of note, treatment of ALCL cell lines with rapamycin also resulted in a slight decrease of Ser⁴⁷³ phosphorylation of AKT. Similarly, silencing mTOR gene expression by mTOR-specific siRNA resulted in decreased levels of activation of mTOR signaling proteins and a decrease of p-AKT (Fig. 4B).

Inhibition of mTOR induces cell cycle arrest in ALK+ ALCL cells. Previous studies have shown that inhibition of PI3K using LY294002 or wortmannin resulted in decreased cell growth and apoptosis of ALCL cells (5). Here, we also used LY294002 to inhibit the PI3K pathway in Karpas 299 and SU-DHL1 cells. Our findings show that treatment of both cell lines with increasing concentrations of LY294002 led to cell cycle arrest as shown by decreased cell cycle S-phase fraction in a dose-dependent manner (Supplementary Fig. S1A).

As shown in Fig. 5A, transient transfection of ALK+ ALCL cells with mTOR siRNA resulted in decreased cell proliferation as assessed by MTS assay. By contrast, no significant changes in cell proliferation were seen following transient transfection with control 4E-BP1 siRNA. Cell cycle studies also showed that inhibition of mTOR function by using rapamycin or inhibition of mTOR expression by using mTOR-specific siRNA induced cell cycle arrest as shown by a dose-dependent decrease of cell cycle S phase (data not shown).

To evaluate the molecular basis underlying cell cycle arrest after inhibition of mTOR, we assessed several cell cycle regulatory proteins after treatment with rapamycin or specific mTOR siRNA. The cyclin-dependent kinase (CDK) inhibitors p21^waf1 and p27^kip1 and underphosphorylated retinoblastoma protein levels were up-regulated and cyclin A levels were decreased in a concentration-dependent manner (Fig. 5B and C).

These results suggest that inhibition of mTOR-raptor complex or silencing mTOR gene expression induced cell cycle arrest at G₁ phase in ALK+ ALCL cells.

Inhibition of mTOR induces apoptosis in ALK+ ALCL cells. As reported previously, inhibition of PI3K resulted in increased apoptosis of ALCL cells (5). Our findings confirm previously published data as treatment of Karpas 299 and SU-DHL1 cells with increasing concentrations of LY294002 led to decreased cell viability and increased apoptosis, as determined by Annexin V staining, in a dose-dependent manner (Supplementary Fig. S1B and C).

To determine if inhibition of mTOR has similar biological effects on ALCL cell lines, we studied cell viability and apoptosis after treatment of Karpas 299 and SU-DHL1 with increasing concentrations of rapamycin and after mTOR-specific siRNA. Both treatments induced a decrease in the number of viable cells. Rapamycin treatment decreased the number of viable cells in a concentration-dependent manner (Fig. 6A). Treatment of Karpas 299 cells with 20 nmol/L rapamycin alone decreased the number of viable cells by approximately one third. Similar results were obtained with SU-DHL1 cells (Supplementary Fig. S2). In addition, we found that treatment with rapamycin sensitized ALK+ ALCL cells to chemotherapy. In our preliminary experiments, using increasing concentrations of doxorubicin, treatment of ALCL cells with 1 μmol/L doxorubicin resulted in no change in cell viability. However, when ALCL cells were treated with 1 μmol/L doxorubicin and increasing concentrations of rapamycin, cell viability was lower compared with treatment with rapamycin alone (data not shown).

Rapamycin treatment also showed a concentration-dependent increase in Annexin V–positive cells, indicating that cell death was secondary to apoptosis (Fig. 6A). Morphologic examination of Hoechst-stained cell preparations at 16 hours after treatment with 20 nmol/L rapamycin revealed that Karpas 299 cells showed morphologic evidence of apoptosis, such as nuclear condensation and fragmentation (Fig. 6A). Similarly, silencing of mTOR also resulted in decreased cell viability and increased Annexin V–positive cells in comparison with control cells and with cells transfected with 4E-BP1-specific siRNA (Fig. 6B).

To study the underlying apoptotic mechanisms, expression of apoptosis regulators was assessed after treatment with rapamycin or specific mTOR siRNA. There was a concentration-dependent decrease in the antiapoptotic proteins BCL-2, BCL-XL, c-FLIP (long and short), and full-length MCL-1 with increasing concentrations of rapamycin (Fig. 6C). Down-regulation of c-FLIP was even more prominent when rapamycin was combined with 1 μmol/L doxorubicin. Similarly, there was decreased expression of c-FLIP, MCL-1, and BCL-2 after silencing of mTOR (Fig. 6D).

These findings suggest that inhibition of mTOR induces apoptosis by modulating the expression of proteins involved in both extrinsic and intrinsic (mitochondrial) apoptotic pathways.

Emerging data indicate that mTOR is frequently activated in many cancers as a result of genetic alterations of the components in the PI3K/AKT network (25, 26). In ALK+ ALCL, the chimeric protein nucleophosmin-ALK has been shown to activate PI3K and its downstream effector AKT (4). However, the activation status of the mTOR pathway has not been investigated in ALK+ ALCL. This is of interest because effective inhibitors of mTOR are currently available and could form the basis of a novel therapeutic strategy.

Here, we show that the mTOR pathway is activated in ALK+ ALCL. We show that Ser²⁴⁴⁸p-mTOR (27, 28) is detected at high levels in ALK+ ALCL tumors and cell lines and that the downstream effectors of mTOR, 4E-BP1, p70S6K, and rpS6 are also frequently phosphorylated in ALK+ ALCL.

We also provide evidence that activation of the mTOR pathway in ALK+ ALCL depends, at least in part, on PI3K/AKT. We observed that the phosphorylation level of AKT significantly correlated with the phosphorylation level of mTOR and rpS6 as well as with expression of total 4E-BP1 and eIF4E in ALK+ ALCL tumors. We showed that forced expression of AKT in ALK+ ALCL cell lines resulted in a substantial increase in the phosphorylation of mTOR, p70S6K, and rpS6. We also show that inhibition of the PI3K/AKT pathway in ALK+ ALCL cell lines decreased the level of phosphorylation of mTOR and mTOR signaling proteins. Our findings agree with the generally accepted model of the PI3K/AKT/mTOR pathway in which PI3K/AKT plays an important upstream positive regulatory role in mTOR activation (6, 29).

PI3K/AKT/mTOR is considered to be a central regulatory pathway of protein translation involved in the regulation of cell proliferation, growth, differentiation, and survival (11, 26, 30). Upon activation, mTOR phosphorylates its downstream targets. p70S6K, a key element of mTOR pathway, promotes cell growth by inducing protein synthesis and cell survival by phosphorylating (inactivating) the proapoptotic protein BAD (31, 32). Activated p70S6K, in part through activation of rpS6 (33), increases the translation of 5′-terminal oligopyrimidine tract mRNAs (31, 34). 5′-Terminal oligopyrimidine tract mRNAs exclusively encode components of the translation machinery, including all ribosomal proteins and elongation factors (34). In addition to the cytoplasmic staining for p-p70S6K, we also detected nuclear staining in a significant proportion of ALK+ ALCL tumors. The presence of p70S6K in the nucleus has been reported in other tumor types (35). In addition, others (36) have shown that inhibition of exportin 1, a nuclear export receptor, can lead to accumulation of p70S6K in the nucleus of kidney cells. However, the function of p70S6K in the nucleus is not clear.

The translation initiation factor, eIF4E, is an indirect target of mTOR because it is released by phosphorylation of its repressor 4E-BP1 by mTOR-raptor complex, thereby stimulating cap-dependent translation (7, 11, 12). Although its role in oncogenesis is poorly understood, expression of eIF4E has been reported in aggressive forms of non-Hodgkin's lymphoma and other cancers (37–39). In agreement with previous studies (39), we observed eIF4E immunoreactivity in all ALCL tumors, with strong expression in almost two thirds of cases. We observed that treatment of ALK+ ALCL cell lines with the PI3K inhibitor, LY294002 (data not shown), and with the mTOR inhibitor, rapamycin, resulted in decreased expression of total eIF4E. It is known that transcription of eIF4E is regulated by MYC and that eIF4E also enhances MYC translation (40). It is plausible that a positive stimulatory loop exists wherein eIF4E stimulates MYC translation, which in turn stimulates eIF4E transcription, thereby perpetuating positive cell growth signals.

The data presented here are intriguing regarding the AKT/mTOR pathway model. Silencing of total mTOR gene expression by siRNA decreased AKT phosphorylation. This finding indicates that mTOR can modulate the activation of AKT and suggests that there is a positive feedback signal to AKT from mTOR (Fig. 7). In agreement with this observation, recently Sarbassov et al. (13) have shown that rictor-mTOR (but not mTOR-raptor) complex directly phosphorylates AKT on Ser⁴⁷³ and facilitates Thr³⁰⁸ phosphorylation by PDK1 in vitro.

Raptor is a 150-kDa mTOR-binding protein that also binds 4E-BP1 and p70S6K (41, 42). Binding of raptor to mTOR is necessary for phosphorylation of 4E-BP1 and p70S6K (Fig. 7). Rapamycin forms an inhibitory complex with the FRB domain of mTOR and inhibits the kinase activities of mTOR protein (18, 19). Our results confirm that rapamycin inhibits activation of the mTOR pathway in ALK+ ALCL cells. Interestingly, we also found that rapamycin decreased the level of Ser²⁴⁴⁸p-mTOR. Chiang and Abraham (43) have recently shown that mTOR phosphorylation at Ser²⁴⁴⁸ is blocked by rapamycin, and this effect is independent of AKT activation status. Moreover, these authors and others recently have shown that mTOR is directly phosphorylated by p70S6 kinase at Ser²⁴⁴⁸ (43, 44). Because treatment with rapamycin also led to decreased AKT phosphorylation in ALK+ ALCL cells in our in vitro study, it is tempting to speculate that an effector protein downstream of mTOR-raptor may contribute directly or indirectly to AKT activation. On theoretical grounds, a good candidate molecule would be p70S6K, because, in aggregate, the recently published findings suggest that there is a positive feedback signal from p70S6K to mTOR (43, 44) and from mTOR to AKT (ref. 13; Fig. 7).

We also show that rapamycin and total mTOR silencing induces cell cycle arrest and apoptosis interfering with proliferation and survival pathways in ALK+ ALCL. Inhibition of mTOR expression or function blocked cell cycle progression from G₁ to S phase associated with modulation of the CDK inhibitors p21^waf1 and p27^kip1. Furthermore, we show that induction of apoptosis following inhibition of mTOR signaling involved both the extrinsic (c-FLIP) and the intrinsic (MCL-1 and BCL-2) apoptotic pathways. Similar effects of rapamycin have been shown in other tumor types. It has been shown that mTOR inhibition by rapamycin arrests cells in G₁ phase of the cell cycle and there is evidence that this inhibitory effect occurs via the inhibition of activation of p70S6K1 and 4E-BP1 (45). In addition, inhibition of mTOR activity by rapamycin induces apoptosis (46). Silencing mTOR expression using siRNA has not been studied previously in hematopoietic malignancies. Several rapamycin derivatives are currently being assessed in the treatment of patients with advanced cancers, including non-Hodgkin's lymphomas, such as mantle cell lymphoma, with good response rates (16). However, many cancer patients treated with rapamycin analogues do not respond and this makes the use of mTOR siRNA an interesting alternative gene therapy approach, at least from the theoretical point of view.

In summary, we have shown that mTOR signaling is frequently activated in ALK+ ALCL and depends, in part, on activation of PI3K/AKT pathway. Inhibition of the mTOR pathway interferes with essential proliferation and survival pathways in ALK+ ALCL and might form the basis for a novel therapeutic strategy.

Grant support: The University of Texas M.D. Anderson Cancer Center institutional research grant (G.Z. Rassidakis).

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 Dr. K. Walsh for providing us with the myrAkt adenoviral vector.