Abstract
Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70–80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3β. Expression of NPM-ALK in 293T cells led to an increase of pS9-GSK3β (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3β levels. Phosphorylation of pS9-GSK3β by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3β substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3β. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3β inhibition. Furthermore, stable knockdown of GSK3β conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS9-GSK3β and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS9-GSK3β and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S9-GSK3β by PI3K/AKT. The subsequent inhibition of GSK3β activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3β as a mediator of NPM-ALK oncogenesis.
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References
Amin HM, Lai R . (2007). Pathobiology of ALK+ anaplastic large-cell lymphoma. Blood 110: 2259–2267.
Bai RY, Dieter P, Peschel C, Morris SW, Duyster J . (1998). Nucleophosmin-anaplastic lymphoma kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes phospholipase C-gamma to mediate its mitogenicity. Mol Cell Biol 18: 6951–6961.
Bischof D, Pulford K, Mason DY, Morris SW . (1997). Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis. Mol Cell Biol 17: 2312–2325.
Boccalatte FE, Voena C, Riganti C, Bosia A, D'Amico L, Riera L et al. (2009). The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL. Blood 113: 2776–2790.
Bostock CJ, Prescott DM, Kirkpatrick JB . (1971). An evaluation of the double thymidine block for synchronizing mammalian cells at the G1-S border. Exp Cell Res 68: 163–168.
Boulares AH, Yakovlev AG, Ivanova V, Stoica BA, Wang G, Iyer S et al. (1999). Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J Biol Chem 274: 22932–22940.
Cohen P, Frame S . (2001). The renaissance of GSK3. Nat Rev Mol Cell Biol 2: 769–776.
Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA . (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785–789.
Diehl JA, Zindy F, Sherr CJ . (1997). Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. Genes Dev 11: 957–972.
Fernandez-Vidal A, Mazars A, Gautier EF, Prevost G, Payrastre B, Manenti S . (2009). Upregulation of the CDC25A phosphatase down-stream of the NPM/ALK oncogene participates to anaplastic large cell lymphoma enhanced proliferation. Cell Cycle 8: 1373–1379.
Frame S, Cohen P, Biondi RM . (2001). A common phosphate-binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol Cell 7: 1321–1327.
Herman SE, Gordon AL, Wagner AJ, Heerema NA, Zhao W, Flynn JM et al. (2010). Phosphatidylinositol 3-kinase-delta inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals. Blood 116: 2078–2088.
Ito M, Zhao N, Zeng Z, Chang CC, Zu Y . (2010). Synergistic growth inhibition of anaplastic large cell lymphoma cells by combining cellular ALK gene silencing and a low dose of the kinase inhibitor U0126. Cancer Gene Ther 17: 633–644.
Iwahara T, Fujimoto J, Wen D, Cupples R, Bucay N, Arakawa T et al. (1997). Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 14: 439–449.
Jinno S, Suto K, Nagata A, Igarashi M, Kanaoka Y, Nojima H et al. (1994). Cdc25A is a novel phosphatase functioning early in the cell cycle. EMBO J 13: 1549–1556.
Jope RS, Johnson GV . (2004). The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci 29: 95–102.
Kang T, Wei Y, Honaker Y, Yamaguchi H, Appella E, Hung MC et al. (2008). GSK-3 beta targets Cdc25A for ubiquitin-mediated proteolysis, and GSK-3 beta inactivation correlates with Cdc25A overproduction in human cancers. Cancer Cell 13: 36–47.
Lannutti BJ, Meadows SA, Herman SE, Kashishian A, Steiner B, Johnson AJ et al. (2011). CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability. Blood 117: 591–594.
Lim MS, Carlson ML, Crockett DK, Fillmore GC, Abbott DR, Elenitoba-Johnson OF et al. (2009). The proteomic signature of NPM/ALK reveals deregulation of multiple cellular pathways. Blood 114: 1585–1595.
Marzec M, Kasprzycka M, Liu X, Raghunath PN, Wlodarski P, Wasik MA . (2006). Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf. Oncogene 26: 813–821.
Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR . (2006). Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol Cell 21: 749–760.
Meijer L, Skaltsounis AL, Magiatis P, Polychronopoulos P, Knockaert M, Leost M et al. (2003). GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 10: 1255–1266.
Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL et al. (1994). Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263: 1281–1284.
Ott GR, Tripathy R, Cheng M, McHugh R, Anzalone AV . (2010). Discovery of a potent inhibitor of anaplastic lymphoma kinase with in vivo antitumor activity. ACS Med Chem Lett 1: 493–498.
Palmer RH, Vernersson E, Grabbe C, Hallberg B . (2009). Anaplastic lymphoma kinase: signalling in development and disease. Biochem J 420: 345–361.
Ruchatz H, Coluccia AM, Stano P, Marchesi E, Gambacorti-Passerini C . (2003). Constitutive activation of Jak2 contributes to proliferation and resistance to apoptosis in NPM/ALK-transformed cells. Exp Hematol 31: 309–315.
Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ et al. (2005). Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23: 94–101.
Slupianek A, Nieborowska-Skorska M, Hoser G, Morrione A, Majewski M, Xue L et al. (2001). Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis. Cancer Res 61: 2194–2199.
van Noort M, Meeldijk J, van der Zee R, Destree O, Clevers H . (2002). Wnt signaling controls the phosphorylation status of beta-catenin. J Biol Chem 277: 17901–17905.
Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C . (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184: 39–51.
Zamo A, Chiarle R, Piva R, Howes J, Fan Y, Chilosi M et al. (2002). Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death. Oncogene 21: 1038–1047.
Zhao Y, Altman BJ, Coloff JL, Herman CE, Jacobs SR, Wieman HL et al. (2007). Glycogen synthase kinase 3alpha and 3beta mediate a glucose-sensitive antiapoptotic signaling pathway to stabilize Mcl-1. Mol Cell Biol 27: 4328–4339.
Acknowledgements
This study was supported in part by funding from the NIH (5R01CA140806-02) awarded to MS Lim, MD/PhD and (R01DE119249 and R01CA136905) KSJ Elenitoba-Johnson, MD. Mangeng Cheng, PhD of Cephalon, Inc provided the small molecule ALK inhibitor.
Author Contributions: SRP McDonnell prepared manuscript, designed and conducted experiments. SR Hwang conducted experiments. V Basrur, PhD and KP Conlon performed mass spectrometry analysis. D Fermin, PhD carried out informatics analysis of mass spectrometry data. E Way, MD and MS Lim, MD/PhD evaluated the results of IHC studies. C Murga-Zamalloa, MD generated SU-DHL-1 and Karpas 299 GSK3β knockdown cells. Z Zeng, PhD and Y Zu, MD generated the SU-DHL-1 cells with tetracycline-inducible shRNA. KSJ Elenitoba-Johnson, MD and MS Lim, MD/PhD designed experiments and prepared manuscript.
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McDonnell, S., Hwang, S., Basrur, V. et al. NPM-ALK signals through glycogen synthase kinase 3β to promote oncogenesis. Oncogene 31, 3733–3740 (2012). https://doi.org/10.1038/onc.2011.542
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DOI: https://doi.org/10.1038/onc.2011.542
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