Oncogenic tyrosine kinase NPM/ALK induces activation of the ... - Nature

Oncogene (2007) 26, 813–821

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ORIGINAL ARTICLE

Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ ERK signaling pathway independently of c-Raf M Marzec, M Kasprzycka, X Liu, PN Raghunath, P Wlodarski and MA Wasik Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALKexpressing T-cell lymphoma (ALK þ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK þ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cyclepromoting CDK4 and phospho-RB. Finally, siRNAmediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies. Oncogene (2007) 26, 813–821. doi:10.1038/sj.onc.1209843; published online 7 August 2006 Keywords: NPM/ALK tyrosine kinase; MEK/ERK pathway; T-cell lymphoma Introduction Under physiologic conditions, ALK tyrosine kinase is expressed only by cells of the nervous system. Its ectopic Correspondence: Professor MA Wasik, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 3400 Spruce Street, 7.106 Founders Pavilion, Philadelphia, PA 191044283, USA. E-mail: [email protected] Received 20 February 2006; revised 2 June 2006; accepted 2 June 2006; published online 7 August 2006

expression in a subset of T-cell lymphomas (ALK þ TCL) and other malignancies typically results from chromosomal translocations involving the ALK gene and various partner genes (Wasik, 2002). The t(2;5) (p23;q35) translocation that occurs in >80% of ALK þ T-cell lymphomas fuses a distal portion of the ALK gene that encodes the entire cytoplasmic region of ALK protein with the proximal part of the nucleophosmin (NPM) gene that encodes the oligomerization domain (Morris et al., 1994; Shiota et al., 1994). The resulting chimeric NPM/ALK protein is constitutively active owing to autophosphorylation and promotes malignant cell transformation as shown both in vitro (Fujimoto et al., 1996; Bischof et al., 1997) and in vivo (Kuefer et al., 1997; Chiarle et al., 2003) by phosphorylating and activating a number of downstream effectors such as Stat3, PI3K, Jak2, Jak3, Grb2, IRS and PLCg (Wasik, 2002). The key mitogen-activated protein kinase (MAPK) signaling pathway results in activation of the extracellular-regulated kinase (ERK) 1 and 2 complexes (Roux and Blenis, 2004). The other, upstream members of the pathway are three related MAPK kinase kinases (MAPKKK) A-Raf (expressed predominantly in urogenital tissue), B-Raf (expressed in neural tissue and testis) and c-Raf (expressed in many cell types including T lymphocytes) and the MAPK kinases (MAPKK) MEK1 and MEK2, which directly activate ERK1 and ERK2 by phosphorylating their key moieties. In turn, the activated ERK1/2 complex activates numerous substrates in all cellular compartments, including various membrane proteins such as CD120a, Syk, cytoskeletal proteins and nuclear substrates SRC-1, Pax6, NF-AT, Elk-1, c-Fos, c-Myc and STAT3 (Hanahan and Weinberg, 2000; Roux and Blenis, 2004). The MEK/ ERK pathway is physiologically activated by growth factors, serum and phorbol esters and, to a lesser degree, cytokines and osmotic stress. The activated MEK/ERK signaling pathway impacts multiple cell functions, including proliferation, survival, migration, division and differentiation (Roux and Blenis, 2004). Aberrant, persistent activation of the MEK/ERK pathway has been found in approximately 30% of human cancers (Hoshino et al., 1999), including cultured and primary cells from the acute and chronic myelogeneous leukemia (Towatari et al., 1997; Kim et al., 1999; Milella et al., 2001; Morgan et al., 2001; Platanias, 2003), natural killer cell large granular lymphocyte

NPM/ALK induces MEK/ERK activation M Marzec et al

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leukemia, multiple myeloma (Hallek et al., 1998; Pratt, 2002; Epling-Burnette et al., 2004) and Epstein–Barr virus (EBV)-transformed B lymphocytes (Wlodarski et al., 2005). It has been demonstrated that the expression of a constitutively active mutant form of either Raf or MEK1 renders hematopoietic cell lines growth factor independent (Tanaka et al., 1996; Hoyle et al., 2000), strongly suggesting that the aberrant, persistent activation of the MEK/ERK pathway plays a key role in malignant transformation of the affected cells. Noteworthy, activation of the MEK/ERK pathway was identified in the cells of neural origin upon expression and activation of the engineered ALK construct (Souttou et al., 2001) and stimulation of the endogeneous, wild-type ALK (Motegi et al., 2004). Here we report that the chimeric, highly oncogenic NPM/ALK tyrosine kinase activates the MEK/ERK pathway and it does so independently of c-Raf. Inhibition of the pathway adversely impacts the proliferation and survival of the NPM/ALK-transformed T cells. Pathogenetic and translational implications of these findings are discussed.

Results Persistent activation of MEK/ERK signaling pathway in cultured and primary ALK þ TCL cells Owing to the key role of MEK/ERK pathway in carcinogenesis (Hoshino et al., 1999; Platanias, 2003), on the one hand, and the reported association of MEK1 and ERK1 with NPM/ALK (Crockett et al., 2004), on the other hand, we examined its activation status in the ALK þ TCL-derived cell lines that preserved key characteristics of the native malignant T cells (Zhang et al., 2002; Marzec et al., 2005). The EBV þ B-cell LCL and EBV BCL cell lines, identified by us as displaying, respectively, activation and lack of activation of the MEK/ERK pathway (Wlodarski et al., 2005), served as controls. As shown in Figure 1a, all four examined ALK þ TCL showed activation of the MEK/ERK pathway as determined by phosphorylation of the MEK1/2 and ERK1/2 complexes. As expected, the control EBV þ BCL cell lines were positive and the EBV–BCL were negative. Notably, the ALK þ TCL and EBV þ B-LCL cell lines also displayed phosphorylation of c-Raf. To ascertain that the activation of MEK/ERK pathway in ALK þ TCL is not limited to the cultured cell lines, we next examined primary lymphoma tissues from 10 ALK þ TCL patients. In all, cases the malignant ALK þ TCL cells strongly expressed phospho-ERK1/2 (representative image is depicted in Figure 1b). Activation of MEK/ERK pathway in ALK þ TCL depends on expression and enzymatic activity of NPM/ ALK Because NPM/ALK is a constitutively self-activated kinase that exerts its cell transforming capacity by activating several proto-oncogenic signaling pathways Oncogene

Figure 1 Activation of the MEK/ERK signaling pathway in ALK þ TCL cells. (a) Western blots with protein lysates of the depicted ALK þ TCL cell lines and control transformed EBVpositive and EBV-negative B-cell lines were performed using antibodies against phospho c-Raf(Ser338), MEK1/2(Ser217/221) and ERK1/2(Thr202/Tyr204). (b) Immunohistochemical analysis of ALK þ TCL tissues. Left panel: histology of a representative case with a large cluster of the malignant cells present in the center. Middle panel: staining with the anti-ALK antibody. Right panel: staining with the anti-phospho-ERK1/2(Th202/Tyr204) antibody. The depicted results are representative of 10 ALK þ TCL cases.

(Wasik, 2002), we next asked if NPM/ALK is responsible for the activation of the MEK/ERK pathway in ALK þ TCL. We employed three different experimental approaches to answer this question. First, we examined the activation of the pathway in the lymphoid, growth factor (interleukin (IL)-3)-dependent BaF3 cell line stably transfected with a vector containing NPM/ALK or an empty vector (EV) as a control. Whereas in the presence of IL-3, both NPM/ALK- and empty vectorcarrying BaF3 cells displayed phosphorylation of c-Raf, MEK1/2 and ERK1/2, only the NPM/ALK-transfected cells maintained the phosphorylation after withdrawal of the cytokine (Figure 2a). These findings indicate that NPM/ALK is, indeed, capable of inducing persistent activation of the pathway. Secondly, to demonstrate that NPM/ALK activates MEK/ERK not only in the transfected but also in the native ALK þ TCL cells, we depleted NPM/ALK by siRNA in the ALK þ TCL-derived Karpas 299 cell line that is amenable to this type of manipulation (Marzec et al., 2005). As shown in Figure 2b, the cells treated with the ALK-specific (but not the control, nonspecific) siRNA displayed a marked decrease in the NPM/ALK expression and, more importantly, resulted in the inhibition of the pathway as determined by ERK1/2 dephosphorylation. The expression of ERK1/2 as well as two other, randomly chosen control proteins STAT3 and S6P remained unaffected by the ALK siRNA, confirming specificity of the depletion. Lastly, to document that the MEK/ERK activation depends on the enzymatic activity of NPM/ALK, we treated ALK þ TCL cells with a small molecule kinase inhibitor, WHI-154, which directly inhibits NPM/ALK


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Figure 2 Suppression of NPM/ALK expression and function inhibits activation of the MEK/ERK pathway in ALK þ TCL cells. (a) Activation of the MEK/ERK pathway in BaF3 cells with enforced expression of NPM/ALK. IL-3 dependent lymphoid BaF3 cell line was stably transfected with NPM/ALK or EV and cultured in the presence ( þ ) or absence () of IL-3 for 1 h. Expression of phospho-c-Raf, -MEK1/2 and -ERK1/2 and total ERK1/2 was analysed by Western blots with the depicted antibodies. NPM/ALK expression in the transfected BaF3 cells was verified with the anti-ALK antibody. (b) siRNA-mediated depletion of NPM/ALK in ALK þ TCL cells. Karpas 299 cells were subjected to double transfection at 0 and 24 h with 100 nM of siRNA specific for NPM/ALK or control, non-targeting siRNA. At 72 h, the cells were harvested and analysed by Western blot with the anti-phospho-ERK1/2 and the other depicted control antibodies. (c) Inhibition of enzymatic activity of NPM/ALK. Three different ALK þ TCL cell lines were treated for 1 h with WHI-154 at 15 mM or the drug’s vehicle. Phosphorylation of c-Raf, MEK1/2 and ERK1/2 was assessed by Western blot. Inhibition of NPM/ALK was confirmed by the abrogation of the ALK Tyr1604 phosphorylation.

(Marzec et al., 2005). As presented in Figure 2c, abrogation of the NPM/ALK function, as demonstrated by the total inhibition of the kinase autophosphorylation, suppressed phosphorylation of c-Raf, MEK1/2 and ERK1/2. The above experiments show that NPM/ ALK induces activation of the MEK/ERK pathway in cells transformed by the kinase. NPM/ALK-induced activation of MEK/ERK signaling pathway is c-Raf independent Because NPM/ALK has been shown to associate directly with MEK1 as well as ERK1 (Crockett et al., 2004), we examined next if activation of c-Raf is required in the ALK þ TCL cells for MEK1/2 and ERK1/2 activation. As shown in Figure 3a (left and middle panels), two separate Raf inhibitors had no effect on ERK1/2 phosphorylation in both examined ALK þ TCL cell lines. This contrasted with the profound effect of the inhibitors on the ERK1/2 phosphorylation seen in the normal, T-cell-rich peripheral blood mononuclear cells (PBMC) that were primed with mitogen (PHA) and stimulated with IL-2 (Figure 3a, right panel). Because protein kinase C (PKC) has been implicated in the MEK/ERK activation in the phorbol ester-stimulated cells, both in a Rafdependent (Ueda et al., 1996; Wang et al., 2004) and -independent (Wen-Sheng, 2006) manner, we next examined if the inhibition of PKC would impact on the MEK/ERK activation in the NPM/ALK-transformed cells. However, two different PKC inhibitors bisindolmaleimide I and Go6983 had no effect on the MEK1/2 and ERK1/2 phosphorylation in the ALK þ

TCL Sudhl-1 cells, although they markedly inhibited cRaf phosphorylation (Figure 3b, left panel). In contrast, the inhibitors all but abrogated phosphorylation of not only c-Raf but also MEK1/2 and ERK1/2 in the normal, phorbol ester (phorbol 12-myristate 13-acetate (PMA))-activated PBMC (Figure 3b, right panel). These results indicate that PKC is not involved in the activation of the MEK/ERK pathway in the NPM/ ALK-transformed T cells. Furthermore, they provide additional evidence that MEK/ERK is activated by NMP/ALK independently of c-Raf. To provide the final, direct evidence that the NPM/ALK-promoted MEK/ERK activation is independent of c-Raf, we depleted c-Raf in Karpas 299 cells with siRNA. This depletion had no effect on ERK1/2 phosphorylation (Figure 3c). It also did not affect cell proliferation and apoptotic rate (data not presented). NPM/ALK-induced ERK activation is MEK dependent To determine if MEK1/2 complex is involved in the activation of ERK1/2 in the ALK þ TCL cells, we treated the cells with the MEK1/2 inhibitor U0126. As shown in Figure 4a, the treatment led to the essentially complete inhibition of the ERK1/2 phosphorylation. Phosphorylation of the control proteins (MEK1/2, ALK and STAT3) was not affected, supporting the specificity of the inhibition. The similar inhibition of ERK1/2 phosphorylation was also identified in the NPM/ALKexpressing, IL-3-depleted BaF3 cells (Figure 4b) exposed to U0126, confirming that MEK1/2 acts as an intermediary in the NPM/ALK-promoted activation of ERK1/2. Oncogene


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Figure 3 Lack of inhibition of MEK/ERK pathway upon inactivation or depletion of c-Raf in ALK þ TCL cells. (a) Lack of inhibition of ERK1/2 phosphorylation by Raf inhibitors. ALK þ TCL-derived Sudhl-1 and Karpas 299 cell lines were treated for 1 h with 2 mM of RI or 0.1 mM of ZM3363372 and evaluated by Western blotting for c-Raf and ERK1/2 phosphorylation and total expression. Mitogen (PHA)-preactivated, normal PBMC treated for 1 h with IL-2 served as a positive control for the inhibition of ERK1/2 by RI and ZM3363372. (b) Lack of inhibition of ERK1/2 phosphorylation by PKC inhibitors. ALK þ TCL-derived Sudhl-1 cell line was treated for 30 min with 1 mM of bisindolmaleimide I and 1 mM of Go6983 and examined by Western blotting for c-Raf, MEK1/2 and ERK1/2 phosphorylation. Phorbol ester (PMA)-preactivated, normal PBMC (PMA Blasts) served as an additional positive control. (c) A lack of effect of c-Raf depletion on ERK1/2 phosphorylation. ALK þ TCL Karpas 299 cells were subjected to double transfection with 100 nM of siRNA specific for c-Raf or non-targeting, control (Ctrl) siRNA. After 48 h, the cells were harvested and analysed by Western blotting with the depicted antibodies. The degree of c-Raf depletion is displayed in the uppermost panel.

Figure 4 Inhibition of MEK enzymatic activity suppresses phosphorylation of ERK1/2 in ALK-expressing cells. (a) MEK inhibitiormediated suppression of ERK1/2 phosphorylation in ALK þ TCL. Sudhl-1, Sup-m2 and Karpas 299 cells were treated for 1 h with 10 mM of U0126, lysed and analysed by Western blotting. Analysis of expression of total ERK1/2, MEK 1/2, ALK and STAT3 as well as phospho-Mek1/2 served as control. (b) MEK inhibitor-mediated suppression of ERK1/2 phosphorylation in NPM/ALKtransfected cells. Lymphoid BaF3 cells transfected with NPM/ALK or EV were cultured without IL-3 for 2 h and treated with 5 mM of U0126 for 1 h. Phosphorylation of ERK1/2 was assessed by Western blot with expression of NPM/ALK and ERK1/2 serving as controls.

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Inactivation of MEK enzymatic activity inhibits proliferation and increases apoptotic cell death of ALK þ TCL cells To determine the biological role of the activated MEK/ ERK pathway in the NPM/ALK-transformed T cells, we examined proliferation and viability of such cells upon their exposure to U0126. As shown in Figure 5a, the MEK inhibitor profoundly suppressed proliferation of the ALK þ TCL cells. The compound also increased the apoptosis of the cells (Figure 5b). The pro-apoptotic effect of U0126 was particularly pronounced when the ALK þ TCL cells were simultaneously treated with etoposide (ET) (Pardo et al., 2002). When combined, the drugs induced apoptotic cell death in approximately 50% of cells, roughly twice as much as any of the drugs used alone (Figure 5c). The pro-apoptotic and antiproliferative effect of U0126 was associated with the negative impact on the respective key proteins involved in these cell functions. As shown in Figure 5d, the drug inhibited the expression of the pro-survival factor Bcl-xL and promoters of cell cycle progression CDK4 and phospho-RB. Depletion of ERK1/2 complex inhibits proliferation and increases apoptosis of ALK þ TCL cells To verify the functional importance of the MEK/ERK pathway in the ALK þ TCL by a different method and

to document that ERK1 and ERK2 are critical mediators of MEK signaling in such cells, we depleted in Karpas 299 cells each of the ERKs by siRNA (Figure 6a). The depletion of either ERK1 or ERK2 markedly diminished cell proliferation; no additional inhibition could be appreciated when ERKs were depleted together (Figure 6b). Notably, whereas the depletion of ERK1 markedly increased cell apoptosis, loss of ERK2 alone had little, if any effect (Figure 6c). Furthermore, when ERK1 and ERK2 were depleted simultaneously, ERK2 loss reproducibly protected cells from the detrimental effect of the ERK1 depletion (Figure 6c).

Discussion It is well established that the chimeric, constitutively self-activated NPM/ALK tyrosine kinase transforms T lymphocytes and other target cells by persistently activating cell signaling pathways. A few NPM/ALKactivated pathways have been identified so far with the key role of STAT3 in the NPM/ALK-mediated oncogenesis being the best characterized (Zhang et al., 2002; Chiarle et al., 2005). A recent study (Crockett et al., 2004) demonstrates, however, that NPM/ALK interacts

Figure 5 Inhibition of MEK enzymatic activity suppresses proliferation and decreases viability of ALK þ TCL cells. ALK þ TCLderived SUDHL-1 and Karpas 299 cell lines were cultured with 10 mM of U0126 and evaluated for BrdU uptake at 24 and 48 h time points (a) and DNA fragmentation at 24 h (b). (c) MEK inhibition augments apoptotic effect of ET. Sudhl-1 cells were cultured for 24 h in the presence of U0126 at 5 mM and/or ET at 4 mM and analysed by flow cytometry following TUNEL staining. Panels a–c depict mean results7s.d. of three independent experiments. (d) MEK inhibition results in the suppression of proteins involved in cell survival and proliferation. ALK þ TCL cell lines Sudhl-1 and Karpas 299 were treated for 24 h with 5 mM of U0126 and analysed by Western blotting for expression of the antiapoptotic Bcl-xL and cell-cycle control proteins CDK4 and phospho-Rb. Total Rb and phosphoERK1/2 and total ERK1/2 served as controls. Oncogene


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Figure 6 Suppression of ALK þ TCL proliferation and viability upon depletion of ERK1 and ERK2. ALK þ TCL Karpas 299 cells were subjected to double transfection with 100 nM of siRNA specific for ERK1 or ERK2, or non-targeting, control siRNA. After 48 h, the cells were harvested and analysed by (a) Western blot with the ERK1 and ERK2 antibodies, (b) EIA to determine cell proliferation by BrdU uptake and (c) flow cytometry following TUNEL staining to determine apoptotic cell rate. The figure shows representative results of three independent experiments with panels b and c depicting mean data7s.d.

with as many as 45 different proteins indicating that the effect of the chimeric kinase on cell signaling is even more profound. In this report, we provide several lines of evidence that NPM/ALK induces activation of MEK1/2 and ERK1/2. This activation is c-Raf independent as documented by both functional inhibition and by the depletion of c-Raf. Activation of ERK1/2 is, however, strictly dependent on the intact activity of MEK1/2. With regard to cell function, the inhibition of the MEK/ERK pathway adversely affects the proliferation and survival of the ALK þ TCL cells. These findings identify MEK/ERK as a new, important signaling pathway in the ALK-induced malignant cell transformation. Our data demonstrate that c-Raf, although activated in the NPM/ALK þ TCL cells, is not required in these Oncogene

cells for activation of MEK1/2 and ERK1/2. Accordingly, inactivation in these cells of its enzymatic activity by two different inhibitors, RI and ZM336372, had no negative effect on the phosphorylation of MEK1/2 and ERK1/2. Similarly, inhibition of c-Raf phosphorylation by PKC inhibitors bisindolmaleimide I and Go6983 also had no effect on the MEK1/2 and ERK1/2 phosphorylation. Finally, siRNA-mediated depletion of c-Raf also did not affect the MEK1/2 and ERK1/2 phosphorylation. These findings suggest that NPM/ALK induces MEK activation directly. The finding that NPM/ALK associates with both MEK1 and ERK1 (Crockett et al., 2004) supports this conclusion. However, given the lack of any evidence that the NPM/ALK may also act as a serine/treonine kinase, it is likely that the MEK1/2 phosphorylation at Ser217/221 identified by us is mediated by another presently unidentified, yet NPM/ ALK-dependent kinase. We also demonstrated that the depletion of either ERK1 or ERK2 comparably impaired cell proliferation but only the depletion of ERK1 significantly increased apoptotic cell rate. Furthermore, ERK2 depletion counteracted the pro-apoptotic effect of the ERK1 loss. This finding is in accordance with the recent observation made in cervical carcinoma HeLa cells in which ERK2 depletion protected the cells from apoptosis induced by double-stranded RNA and other stimuli (Ussar and Voss, 2004). Taken together, these findings indicate that ERK2 exerts a pro- rather than an antiapoptotic effect, at least in certain types of cells. Notably, simultaneous inactivation, rather than the depletion of ERK1 and ERK2 by the MEK1/2 inhibitor, resulted in cell apoptosis. Taken together, these observations indicate that functional consequences of depletion vs inhibition of ERK2 and, for that matter, any other kinase may be quite different. In more general terms, the ERK depletion experiments indicate that, similar to MEK1 and MEK 2 (Matsumoto et al., 2005), ERK1 and ERK2 display some distinct functions. Given the large number of proteins identified as substrates of the ERK1/2 complex (Hanahan and Weinberg, 2000; Roux and Blenis, 2004), it is likely that some of the proteins interact only with one of the ERKs. Further studies are, therefore, required to determine the respective contribution of ERK1 and ERK2 to cell signaling. Our findings that the MEK/ERK pathway stimulates cell proliferation and overall enhances cell viability indicate that members of the pathway represent potential, novel therapeutic targets in the ALK-driven tumors. Although recent data demonstrate that NPM/ ALK itself is an attractive therapeutic target for small molecule inhibitors (Marzec et al., 2005; Wan et al., 2006), it could be argued that a rationally designed combination of an ALK inhibitor with inhibitor(s) of its key effectors such as MEK1/2 and/or ERK1/2 might have an even more pronounced therapeutic effect. However, the lack of clinical quality inhibitors of NPM/ALK as well as MEK1/2, ERK1/2 and other NPM/ALK-activated signal transducers currently prevents the development of such combinations.


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In summary, we demonstrate that NPM/ALK activates MEK1/2 and its direct target ERK1/2 independently of c-Raf and that the MEK/ERK pathway promotes proliferation and survival of the ALK þ TCL cells. These findings identify MEK/ERK as a novel aberrantly activated, and biologically important signaling pathway in ALK þ TCL and suggest that the pathway represents a potential therapeutic target in ALK-driven malignancies.

Materials and methods ALK þ TCL, B-cell and BaF3 cell lines and normal activated lymphocytes Sudhl-1, Sup-m2, Jb6 and Karpas 299 cell lines were derived from four different CD30 þ anaplastic large T-/null-cell lymphomas and carry the t(2;5) chromosomal translocation involving ALK and NPM genes as documented by cytogenetic, molecular and protein expression analysis (Zhang et al., 2002; Marzec et al., 2005). EBV-positive lymphoblastoid B-cell lines (B-LCL) MM and HH were established in our laboratory by the EBV-mediated immortalization of peripheral blood B lymphocytes (Wlodarski et al., 2005). Val and Ly18 cell lines were derived from the EBV-negative diffuse large B-cell lymphoma (BCL; 21). The murine pro-B-cell lymphoid cell line BaF3 was transfected with a vector containing NPM/ALK construct or a control, EV (Zhang et al., 2002; Marzec et al., 2005). Normal activated lymphocytes were obtained by either mitogen (PHA) stimulation of PBMC (PHA Blasts) for 6 days following 1 h stimulation with IL2 or 50 ng/ml of PMA (Sigma-Aldrich, St Louis, MO, USA) for 24 h. The cell populations were cultured at 371C and 5% CO2 in standard RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin/fungizone mixture, 2 mM L-glutamine and, in the case of BaF3 cells, 10% of IL-3 containing WEHI-conditioned medium or recombinant IL-3. Kinase inhibitors U0126 (Promega, Madison, WI, USA) is an MEK1/2 inhibitor and was used at the concentrations of 5–15 mM. WHI-154 (Calbiochem/EMD Biosciences, La Jolla, CA, USA), previously described by us as a potent ALK inhibitor, was used at a concentration of 15 mM. ZM336372 (Calbiochem) is a c-Raf inhibitor and, to a lesser extent, also a B-Raf inhibitor and was used at a concentration of 0.1 mM. Raf1 kinase inhibitor I (designed RI: Calbiochem) was used at the concentration of 2 mM. Bisindolmaleimide I and Go6983 (Calbiochem) are broad-specificity PKC inhibitors and were used at 1 mM. Western blot The cells were washed briefly in phosphate-buffered saline (PBS), centrifuged and lysed in an RIPA buffer (50 mM TrisHCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA)) supplemented with 0.5 mM phenylmethylsulphonyl flouride, phosphatase inhibitor cocktails I and II from Sigma and protease inhibitor cocktail from Roche (Basel, Switzerland), according to the manufacturer’s specifications. For normalization of the gel loading, the protein extracts were assayed with Lowry’s method (Bio-Rad Dc protein assay). Typically, 20–30 mg of the protein per lane was loaded. To examine protein phosphorylation, the membranes were incubated with the antibodies (from Cell Signaling, Boston, MA, USA) specific

for c-Raf Ser338, MEK1/2 Ser217/221, ERK1/2 Thr202/ Tyr204 or ALK phosphorylated on tyrosine positioned at 1604 in the nonchimeric, native protein. To detect total protein, we used c-Raf, MEK1/2, ERK1/2, S6P antibodies (Cell Signaling) and STAT3 and ALK antibodies (Santa Cruz, Santa Cruz, CA, USA). Next, the membranes were incubated with the appropriate secondary, peroxidase-conjugated antibodies. The blots were developed using the SuperSignal West Dura from Pierce (Rockford, IL, USA). Immunohistochemical analysis The analysis was carried out using the anti-phospho(p)-ERK1/ 2(Tyr202/204) antibody from Cell Signaling and the ENVISION horseradish peroxidase polymer method (Dako, Glostrup, Denmark). Formalin-fixed paraffin-embedded tissue specimens were deparaffinized and heat induced for antigen retrieval by boiling the slides in 10 mM citrate buffer (pH 6) for 20 min. The sections were blocked for 10 min with peroxidase blocking system and incubated at room temperature with the anti-p-ERK1/2 rabbit polyclonal primary antibody at 1:50 dilution for 90 min and secondary anti-rabbit IgG horseradish peroxidase polymer for 30 min. After having been washed, the slides were exposed to the chromagen DABplus from Dako for 5 min and counterstained with hematoxylin. siRNA assay A mixture of four c-Raf, or ERK1, or ERK2, or ALK-specific siRNA or nonsense siRNA (all purchased from Dharmacon, Chicago, IL, USA) was introduced into cells at 100 nM by lipofection with the new generation Lipofectamine (DMRIE-C; Invitrogen, Carlsbad, CA, USA). The procedure was repeated after 24 h. The cells were harvested at two time points (48 h for c-Raf and ERK1/2 siRNA experiments and 72 h for NPM/ALK siRNA experiment) after the first transfection. The extent of the protein knockdown was examined by Western blotting. In vitro cell proliferation (BrdU incorporation) assay After the cell culture for 24 h with the U0126 inhibitor at the concentration of 10 mM, or 48 h with 100 nM of siRNA, cell proliferation was evaluated by detection of 50 -bromo-20 deoxyuridine (BrdU) incorporation using the commercially available kit Cell Proliferation ELISA (Roche) according to the manufacturer’s protocol. In brief, cells were seeded in 96well plates (Corning) at a concentration of 1 104 cells/well in RPMI medium supplemented with 10% FBS and labeled with BrdU (Roche) for 4 h. After the plate centrifugation (10 min at 300 g), supernatant removal and plate drying, the cells were fixed and the DNA was denaturated by adding 200 ml FixDenat reagent. The amount of incorporated BrdU was determined by incubation with a specific antibody conjugated with peroxidase followed by colorimetric conversion of the substrate and OD evaluation in the ELISA plate reader. Cell apoptosis (TUNEL) assay We used the ApoAlert DNA Fragmentation Assay Kit from BD Bioscience (San Jose, CA, USA) according to the manufacturer’s protocol. In brief, after the cell culture at 0.5 106 cells/ml for 24 h with the U0126 inhibitor at a concentration of 10 mM and, in some samples, ET (SigmaAldrich, St Louis, MO, USA) at the dose of 4 mM or for 48 h with 100 nM of siRNA, the cells were collected, washed twice in PBS and fixed with 1% formaldehyde/PBS. After the wash, the cells were permeabilized with 70% ice-cold ethanol for at least 2 h, washed and incubated in TdT incubation buffer for 1 h at 371C. The reaction was stopped by adding 20 mM EDTA and Oncogene


820 the cells were washed twice in 0.1% Triton X-100/BSA/PBS. Finally, samples were resuspended in 0.5 ml of PI/RNAse/ PBS, then collected and analysed by Flow Cytometry (FACSort BD) using the CellQuest PRO software.

Acknowledgements This work was supported in part by grants from the National Cancer Institute – R01-CA89194 and R01-CA96856.

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