Multilevel Dysregulation of STAT3 Activation in Anaplastic Lymphoma Kinase-Positive T/Null-Cell Lymphoma 1 Qian Zhang,* Puthryaveett N. Raghunath,* Liquan Xue, ‡ Miroslaw Majewski,* David F. Carpentieri, †¶ Niels Odum, § Stephan Morris, ‡ Tomasz Skorski, †¶ and Mariusz A. Wasik 2 * 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 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 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. I t has been proposed recently that expression of anaplastic lymphoma kinase (ALK) 3 defines a distinct clinicopatholo- logical entity within the non-Hodgkin’s lymphoma category (1–3). The ALK + lymphomas occur predominantly in children and young adults as a disseminated disease with frequent involvement of extranodal sites. ALK + lymphomas usually display an anaplas- tic large cell morphology and express CD30 Ag (reviewed in Refs. 4 and 5). They almost invariably express a T/null-cell phenotype, although rare cases of ALK + B cell lymphoma have also been described (6). The expression of ALK represents an independent, favorable prognostic factor in all patient age groups (1, 7, 8). How- ever, other variables such as high stage of the lymphoma (7) and the presence of activated cytotoxic T lymphocytes (9) negatively impact on the treatment outcome. Most studies have demonstrated that ALK expression is useful in distinguishing ALK + lymphoma from other entities such as Hodgkin lymphoma (10 –20) and pri- mary cutaneous CD30 + lymphoproliferative disorders (15, 17, 18, 21, 22), particularly when detected on the protein level by immu- nohistochemical staining. Expression of ALK, which is physiologically confined to cells of the nervous system (23), results in lymphomas from at least five different translocations involving ALK gene locus on the short arm of chromosome 2 (24 –33). The most frequent is a translocation t(2;5) (p23;q35) that occurs in up to 80% of cases of the ALK + T/null-cell lymphoma (TCL). It fuses the ALK gene with a gene 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- kDa NPM/ALK chimeric protein contains the oligomerization motif of NPM fused to the cytoplasmic portion of ALK that in- cludes an intact kinase catalytic domain (38 – 40). NPM/ALK is constitutively activated through tyrosine autophosphorylation (41, 42). It has cell-transforming properties as demonstrated in in vitro (41– 43) and in vivo (44, 45) experimental systems. However, the mechanisms of NPM/ALK-mediated oncogenesis remain poorly defined (41, 45, 46). STATs are members of the ubiquitously expressed family of transcription factors activated in response to growth factors and cytokines (reviewed in Refs. 47– 49). Activation of STATs re- quires phosphorylation of their tyrosine residues either by the re- ceptors that often display an intrinsic tyrosine kinase activity or by receptor-associated Jak/Tyk kinases. The phosphorylated STATs form dimers that translocate into the nucleus and initiate transcrip- tion of the growth factor/cytokine-responsive genes. STATs play a critical role in promoting cell proliferation and cell protection from *Department of Pathology and Laboratory Medicine and † Children’s Hospital of Phil- adelphia, University of Pennsylvania, Philadelphia, PA 19104; ‡ Department of Pa- thology, St. Jude Children’s Research Hospital, Memphis, TN 38105; § Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark; and ¶ Center for Biotechnology, Temple University, Philadelphia, PA 19122 Received for publication December 1, 2000. Accepted for publication October 22, 2001. 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 This work was supported in part by National Cancer Institute Grant CA89194 (to 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 the Leukemia and Lymphoma Society. 2 Address correspondence and reprint requests to Dr. Mariusz A. Wasik, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 7.103 Founders, Philadelphia, PA 19104. E-mail address: wasik@mail.med.upenn.edu 3 Abbreviations used in this paper: ALK, anaplastic lymphoma kinase; TCL, T/null- cell lymphoma; NPM, nucleophosmin; PP2A, protein phosphatase A2; PIAS3, pro- tein inhibitor of activated STAT3;  c , common -chain. 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