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T/Null-Cell Lymphoma in Anaplastic Lymphoma Kinase-Positive

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T/Null-Cell Lymphoma in Anaplastic Lymphoma Kinase-Positive Multilevel Dysregulation of STAT3 Activation in Anaplastic Lymphoma Kinase-Positive T/Null-Cell Lymphoma This information is current as Qian Zhang, Puthryaveett N. Raghunath, Liquan Xue, of September 24, 2021. Miroslaw Majewski, David F. Carpentieri, Niels Odum, Stephan Morris, Tomasz Skorski and Mariusz A. Wasik J Immunol 2002; 168:466-474; ; doi: 10.4049/jimmunol.168.1.466 http://www.jimmunol.org/content/168/1/466 Downloaded from References This article cites 88 articles, 51 of which you can access for free at: http://www.jimmunol.org/content/168/1/466.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Multilevel Dysregulation of STAT3 Activation in Anaplastic Lymphoma Kinase-Positive T/Null-Cell Lymphoma1 Qian Zhang,* Puthryaveett N. Raghunath,* Liquan Xue,‡ Miroslaw Majewski,* David F. Carpentieri,†¶ Niels Odum,§ Stephan Morris,‡ Tomasz Skorski,†¶ and Mariusz A. Wasik2* Accumulating evidence indicates that expression of anaplastic lymphoma kinase (ALK), typically due to t(2;5) translocation, defines a distinct type of T/null-cell lymphoma (TCL). The resulting nucleophosmin (NPM) /ALK chimeric kinase is constitutively active and oncogenic. Downstream effector molecules triggered by NPM/ALK remain, however, largely unidentified. Here we report that NPM/ALK induces continuous activation of STAT3. STAT3 displayed tyrosine phosphorylation and DNA binding in all (four of four) ALK؉ TCL cell lines tested. The activation of STAT3 was selective because none of the other known STATs was Downloaded from consistently tyrosine phosphorylated in these cell lines. In addition, malignant cells in tissue sections from all (10 of 10) ALK؉ TCL patients expressed tyrosine-phosphorylated STAT3. Transfection of BaF3 cells with NPM/ALK resulted in tyrosine phosphory- lation of STAT3. Furthermore, STAT3 was constitutively associated with NPM/ALK in the ALK؉ TCL cell lines. Additional studies into the mechanisms of STAT3 activation revealed that the ALK؉ TCL cells expressed a positive regulator of STAT3 activation, protein phosphatase 2A (PP2A), which was constitutively associated with STAT3. Treatment with the PP2A inhibitor calyculin A abrogated tyrosine phosphorylation of STAT3. Finally, ALK؉ T cells failed to express a negative regulator of activated http://www.jimmunol.org/ STAT3, protein inhibitor of activated STAT3. These data indicate that NPM/ALK activates STAT3 and that PP2A and lack of protein inhibitor of activated STAT3 may be important in maintaining STAT3 in the activated state in the ALK؉ TCL cells. These results also suggest that activated STAT3, which is known to display oncogenic properties, as well as its regulatory molecules may .represent attractive targets for novel therapies in ALK؉ TCL. The Journal of Immunology, 2002, 168: 466–474 t has been proposed recently that expression of anaplastic impact on the treatment outcome. Most studies have demonstrated lymphoma kinase (ALK)3 defines a distinct clinicopatholo- that ALK expression is useful in distinguishing ALKϩ lymphoma logical entity within the non-Hodgkin’s lymphoma category from other entities such as Hodgkin lymphoma (10–20) and pri- ϩ ϩ I by guest on September 24, 2021 (1–3). The ALK lymphomas occur predominantly in children and mary cutaneous CD30 lymphoproliferative disorders (15, 17, 18, young adults as a disseminated disease with frequent involvement 21, 22), particularly when detected on the protein level by immu- ϩ of extranodal sites. ALK lymphomas usually display an anaplas- nohistochemical staining. tic large cell morphology and express CD30 Ag (reviewed in Refs. Expression of ALK, which is physiologically confined to cells 4 and 5). They almost invariably express a T/null-cell phenotype, of the nervous system (23), results in lymphomas from at least five ϩ although rare cases of ALK B cell lymphoma have also been different translocations involving ALK gene locus on the short arm described (6). The expression of ALK represents an independent, of chromosome 2 (24–33). The most frequent is a translocation favorable prognostic factor in all patient age groups (1, 7, 8). How- t(2;5) (p23;q35) that occurs in up to 80% of cases of the ALKϩ ever, other variables such as high stage of the lymphoma (7) and T/null-cell lymphoma (TCL). It fuses the ALK gene with a gene the presence of activated cytotoxic T lymphocytes (9) negatively encoding nucleophosmin (NPM), which is a ubiquitously ex- pressed protein involved in shuttling of ribosomal components be- tween the cytoplasm and the nucleus (34–37). The resulting 80- *Department of Pathology and Laboratory Medicine and †Children’s Hospital of Phil- kDa NPM/ALK chimeric protein contains the oligomerization adelphia, University of Pennsylvania, Philadelphia, PA 19104; ‡Department of Pa- thology, St. Jude Children’s Research Hospital, Memphis, TN 38105; §Institute of motif of NPM fused to the cytoplasmic portion of ALK that in- Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, cludes an intact kinase catalytic domain (38–40). NPM/ALK is ¶ Denmark; and Center for Biotechnology, Temple University, Philadelphia, PA constitutively activated through tyrosine autophosphorylation (41, 19122 42). It has cell-transforming properties as demonstrated in in vitro Received for publication December 1, 2000. Accepted for publication October 22, 2001. (41–43) and in vivo (44, 45) experimental systems. However, the The costs of publication of this article were defrayed in part by the payment of page mechanisms of NPM/ALK-mediated oncogenesis remain poorly charges. This article must therefore be hereby marked advertisement in accordance defined (41, 45, 46). with 18 U.S.C. Section 1734 solely to indicate this fact. STATs are members of the ubiquitously expressed family of 1 This work was supported in part by National Cancer Institute Grant CA89194 (to transcription factors activated in response to growth factors and M.A.W.) and American Cancer Society Grant RPG9834601LBC (to T.S.). M.A.W. is a recipient of the National Cancer Institute Shannon Award and T.S. is a Scholar of cytokines (reviewed in Refs. 47–49). Activation of STATs re- the Leukemia and Lymphoma Society. quires phosphorylation of their tyrosine residues either by the re- 2 Address correspondence and reprint requests to Dr. Mariusz A. Wasik, Department of ceptors that often display an intrinsic tyrosine kinase activity or by Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 7.103 receptor-associated Jak/Tyk kinases. The phosphorylated STATs Founders, Philadelphia, PA 19104. E-mail address: [email protected] form dimers that translocate into the nucleus and initiate transcrip- 3 Abbreviations used in this paper: ALK, anaplastic lymphoma kinase; TCL, T/null- cell lymphoma; NPM, nucleophosmin; PP2A, protein phosphatase A2; PIAS3, pro- tion of the growth factor/cytokine-responsive genes. STATs play a ␥ ␥ tein inhibitor of activated STAT3; c, common -chain. critical role in promoting cell proliferation and cell protection from Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 The Journal of Immunology 467 apoptosis. Several members of the STAT family have been iden- pared with activity of two different concentrations (2 U and 4 U) of a tified and designated STAT1 to STAT6. STAT5 comprises two purified human epidermal growth factor receptor supplied by the kit’s closely related but distinct STATs labeled STAT5a and STAT5b. manufacturer. ␤ STAT1 and STAT3 have two isoforms (STAT1 and STAT3 ) Protein expression and phosphorylation resulting from an alternative gene splicing. These experiments were performed as described previously (54–56). In Here we show that NPM/ALK induces constitutive activation of 6 ϩ brief, the cells (5–10 ϫ 10 ) under study were washed and lysed in ice-cold STAT3 in ALK TCL cells. This activation was present in all ϩ lysis buffer. In some experiments, the cells were exposed for 5 min to 500 ALK TCL cell lines (4 of 4) and tissues (10 of 10) tested. STAT3 U of IL-2 or medium alone before lysis. The cell lysates were centrifuged, activation was selective because the other six STATs were incon- and supernatants were precleared with protein A-Sepharose (Sigma-Ald- sistently activated in the ALKϩ TCL cell lines. In addition, STAT3 rich) and incubated with a primary Ab specific for ALK, Jak3, STAT 1–6, became phosphorylated in BaF3 lymphoid cells after transfection PP2A, or PIAS1/3 (all Abs from Santa Cruz Biotechnology) and protein A-Sepharose. In some experiments, Abs were preincubated with a specific with NPM/ALK, but not with an empty vector. Furthermore, inhibitory peptide (54). The immunoprecipitates were washed, boiled,
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