Transcript
Page 1: A New Variant Anaplastic Lymphoma Kinase (ALK)-Fusion Protein … · GTGTG-39); and (e) AP1 and AP2 (from the RACE kit). The 750-bp DNA fragment obtained by nested PCR (see “Results”)

[CANCER RESEARCH 60, 793–798, February 15, 2000]

Advances in Brief

A New Variant Anaplastic Lymphoma Kinase (ALK)-Fusion Protein (ATIC-ALK)in a Case of ALK-positive Anaplastic Large Cell Lymphoma1

Mirella Trinei, Luisa Lanfrancone, Elias Campo, Karen Pulford, David Y. Mason, Pier-Giuseppe Pelicci,2 andBrunangelo FaliniDepartment of Experimental Oncology, Istituto Europeo di Oncologia, 20141 Milan, Italy [M. T., L. L., P-G. P.]; Department of Pathology, University of Barcelona, Barcelona,Spain [E. C.]; LRF Immunodiagnostics Unit, John Radcliffe Hospital, Oxford, United Kingdom [K. P., D. Y. M.]; and Institute of Hematology, University of Perugia, 06100Perugia, Italy [B. F.]

Abstract

Anaplastic lymphoma kinase (ALK)-positive lymphomas (“ALKomas”)constitute a distinct molecular and clinicopathological entity within theheterogeneous group of CD30-positive large cell lymphomas. In 80–85%of cases tumor cells express aMr 80,000 hybrid protein comprising thenucleolar phosphoprotein nucleophosmin (NPM) and the ALK. We reporthere the cloning and expression of a novel ALK-fusion protein from anALK-positive lymphoma. This case was selected for molecular investiga-tion because of (a) the absence of NPM-ALK transcripts; (b) the atypicalstaining patterns for ALK (cytoplasm-restricted) and for NPM (nucleus-restricted); and (c) the presence of aMr 96,000 ALK-protein differing insize from NPM-ALK. Nucleotide sequence analysis of ALK transcriptsisolated by 5*-rapid amplification of cDNA ends revealed a chimericmRNA corresponding to an ATIC-ALK in-frame fusion. ATIC is a bi-functional enzyme (5-aminoimidazole-4-carboxamide ribonucleotidetransformylase and IMP cyclohydrolase enzymatic activities) that cata-lyzes the penultimate and final enzymatic activities of the purine nucleo-tide synthesis pathway. Expression of full-length ATIC-ALK cDNA inmouse fibroblasts revealed that the fusion protein (a) possesses constitu-tive tyrosine kinase activity; (b) forms stable complexes with the signalingproteins Grb2 and Shc; (c) induces tyrosine-phosphorylation of Shc; and(d) provokes oncogenic transformation. These findings point to fusion withATIC as an alternative mechanism of ALK activation.

Introduction

Inappropriate expression of the neural-associated receptor tyrosinekinase ALK3 ( 1,2) defines a distinct clinicopathological entity (ALK-positive lymphomas or “ALKomas”; Refs. (ref3, 4) within the heter-ogeneous group of ALCLs (5–9). In 80–85% of the cases, ALKexpression is the consequence of the t(2;5)(p23;q35) chromosomaltranslocation (10), whose breakpoints are located in theNPMandALKgenes (10, 11). The resultingNPM/ALK chimeric gene encodes aMr

80,000 fusion protein containing the NH2-terminal portion of NPMand the cytoplasmic domain of ALK (11–13). NPM is ubiquitouslyexpressed and encodes a nucleolar protein involved in nucleocyto-plasmic trafficking (14). Similarly to other receptor tyrosine kinase(RTK) oncogenes, fusion with NPM results in constitutive dimeriza-tion and activation of the ALK tyrosine kinase (15).

In about 15% of ALK-positive lymphomas, immunostaining forALK and for NPM shows atypical intracellular localization patterns,and Western blotting analysis of these cases has revealed the presenceof ALK proteins with molecular weights differing from those of ALKand NPM-ALK (16). This correlates with the finding that, in about15–20% of ALK-lymphomas, the t(2;5) is undetectable, and ALKexpression is probably due to the fusion of ALK with other partnergenes. Indeed, rearrangements involving chromosome 2p23 in ALCLshave been recently found in the t(1;2), t(2;3), and inv (2) (17–19); andthe non-muscle tropomyosinTPM3gene (20) and theTFG (21) havebeen cloned as partners of ALK in cases of ALCL carrying, respec-tively, the (1;2) (q25;p23) and (2;3) (p23;q21) translocations.

The cloning of novel ALK partners in ALCL is crucial to theunderstanding of the genetics of ALCLs and the functioning ofALK-fusion proteins. We report here the identification of the ATIC(Pur H) gene as a novel ALK partner in one case of ALCL.

Materials and Methods

Patient. An 18-year old male patient presented in July 1997 with a 4-weekhistory of fever and prominent lymphadenopathy in the neck and the rightinguinal regions. Biopsy of the inguinal lymph node was diagnostic of CD30-positive ALCL of T type (CD31, CD20-, CD79a-, EMA1, CD68-, highfraction of Ki-671 tumor cells; Ref. 6). An attempt to perform cytogeneticanalysis on lymph node cell suspension failed because of the low number andpoor viability of the recovered tumor cells. The disease was staged as IIIB andthe patient was treated with combined chemotherapy (MACOP-B regimen). Atthe last follow-up (June 1999), the patient was in complete remission.

Immunohistochemical Detection of ALK and NPM Proteins. Immuno-histological detection of ALK and NPM protein was performed on lymph nodeparaffin sections using monoclonal antibodies directed against fixative-resis-tant epitopes of the cytoplasmic portion of ALK (antibodies ALK1 and ALKc;Refs. 4, 19), and against the NPM NH2 terminus (antibodies NA24 and NPMa)and COOH-terminus (antibody NPMc; Ref. 22). Before immunostaining,paraffin sections were subjected to antigen retrieval by microwave heating(750 w3 3 cycles of 5 min each) in 1 mM EDTA buffer (pH 8.0; Ref. 4, 22).The immuno-alkaline phosphatase (APAAP) technique was used as the im-munodetection system (4, 22).

RNA Extraction and RACE. Total RNA was prepared from frozen lymphnode samples by a single-step RNA isolation method (TRIzol Reagent, LifeTechnologies). Poly(A)1 RNA was purified from total RNA using oligo (dT)cellulose (MessageMaker Reagent Assembly, Life Technologies). The ATIC-ALK fusion transcript was isolated by 59-RACE, a PCR-based method thatallows rapid amplification of a given cDNA using 39-specific primers (23). The59-RACE from poly(A)1 RNA was performed using the Marathon cDNAAmplification kit (Clontech). The following oligo-primers were used: (a)ALK1 (59-TCCTTGGGCCTCACAGGCACTTTCT39); (b) ALK2(59GGTC-TCTCGGAGGAAGGACTT-GAGGTCT-39; (c) ALK3 (59-TCCTCCTGGT-GCTTCCGGCGGTA-39); (d) ATIC1 (59-CACGCTCGAGTGACAGTGGT-GTGTG-39); and (e) AP1 and AP2 (from the RACE kit). The 750-bp DNAfragment obtained by nested PCR (see “Results”) was cloned into the pTA-dvvector (Clontech) and sequenced by the di-deoxy method. Nucleotide anddeduced aa sequences were subjected to homology search with GenBank using

Received 8/16/99; accepted 1/3/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Associazione Italiana per la Ricerca sul Cancro and the LeukaemiaResearch Fund of Great Britain.

2 To whom requests for reprints should be addressed, at Department of ExperimentalOncology, Istituto Europeo di Oncologia, Via ripamonti, 435, 20141 Milano, Italy.E-mail: [email protected].

3 The abbreviations used are: ATIC, AICAR formal transferase/IMP cyclohydrolase;ALK, anaplastic lymphoma kinase; ALCL, anaplastic large cell lymphoma; NPM, nu-cleophosmin;TFG, TRK-fused gene; RACE, rapid amplification of cDNA ends; aa,amino acid; AICARFT, 5-aminoimidazole-4-carboxamide ribonucleotide transformylase;IMP, inosine 59-monophosphate; IMPCH, IMP cyclohydrolase; EtBr, ethidium bromide;FAICAR, 5-phosphorybosyl-4-carboxamide-5-formamidinomidazole.

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the BLAST search program. The NPM-ALK (22) and ATIC-ALK cDNAswere subcloned into the pCDNA3 expression vectors.

Immunoblotting and Immunoprecipitation. For the preparation of cel-lular lysates from patient samples, cryostat sections (6-um) were cut from aportion of the patient’s lymph node biopsy that had been previously snap-frozen in liquid nitrogen. Sections were air-dried for 5 min, wrapped inaluminum foil, and stored at270°C. Aliquots (50-ml) of sample buffercontaining DTT (Sigma Chemical Co.), were added to each tissue section aftertheir removal from270°C storage. After 5 min at room temperature, the bufferwas aspirated from the slides, heated at 95°C for 4 min, and loaded onto 7.5%SDS-PAGE gels. Cultured cells were lysed in PLC lysis buffer, 50 mM HEPES(pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM

EGTA, 100 mM NaF, 500 mM sodium vanadate, 1 mM phenylmethylsulfonylfluoride, 10 mg/ml aprotinin and 10 mg/ml leupeptin. Immune complexes wereabsorbed on protein G-Sepharose beads (Pharmacia) and washed extensivelywith PLC buffer. For immunoblotting, the proteins were subjected to PAGE-SDS and then transferred to PVDF membrane (Pall). The blotted membranewas probed with appropriate antibodies followed by chemiluminescent detec-tion (Amersham). Monoclonal anti-Alk and anti-Npm antibodies and poly-clonal anti-Shc antibodies were prepared in the authors’ laboratories (4, 19, 22,24). Polyclonal anti-Grb2 and monoclonal antiphosphotyrosine antibodieswere purchased from Santa Cruz Biotechnology and Upstate Biotechnology,respectively.

Focus-forming Assay for Transformation. NIH-3T3 cells were culturedin DMEM/5% bovine calf serum. Cells were seeded onto plastic dishes(1.3 3 105 cells per dish) and transfected with 10mg of plasmid DNA. After24 h, DMEM/5% bovine calf serum was added and scored for focus formationafter 10–14 days by staining with GIEMSA solution.

Results

Identification of ALK-Lymphoma Showing Atypical NPM andALK Localization. The typical intracellular localization pattern ofNPM-ALK in lymphomas carrying the t(2;5), as revealed by theavailable anti-Alk antibodies, is nuclear, with or without cytoplasmicstaining. Immunolabeling with antibodies directed against the NH2-region of NPM, which is retained within the fusion protein, revealssimilar staining patterns (22, 25). We have screened a large panel ofALCLs using monoclonal anti-Alk and anti-Npm antibodies for atyp-ical staining patterns and identified a case in which lymph nodeparaffin sections showed a cytoplasm-restricted expression of ALKand a nucleus-restricted positivity for NPM (Fig. 1). This immuno-histological finding suggested the presence of an ALK- fusion proteinother than NPM-ALK (25).

Cloning of the ATIC-ALK Fusion Transcript. To clone the 59end of the putative novel ALK-fusion cDNA, 59-RACE was per-formed using poly(A)1 RNA from diagnostic and normal lymphnodes. The first PCR, using primers AP1 and ALK1 (Fig. 2A, upperdiagram), enriched for DNA fragments in the diagnostic sample thatwere absent from the control sample (Fig. 2B, Lanes 4–5and 6–7,respectively). Southern blot analysis using the internal primer ALK3as probe (Fig. 2A,upper diagram) confirmed the specificity of thosefragments (data not shown). A 1.3-kb ALK–hybridizing fragment wasgel-purified and used as template in a nested PCR reaction using theAP2 and ALK2 primers (Fig. 2A, upper diagram). The resulting750-bp DNA fragment (Fig. 2C,Lane 2) was purified and cloned intothe pT-Adv vector. DNA sequence analysis revealed a single openreading frame from nucleotides 1 to 735 (245 aas; Fig. 2E). Adatabase search revealed identity (at both nucleotide and proteinlevels) as ATIC (from nucleotides 1 to 274; 26–27) and ALK (fromnucleotides 275 to 735; Refs. 1, 2; Fig. 2E). To confirm the existenceof the ATIC-ALK fusion transcript in the original RNA sample fromthe biopsy, a PCR reaction was performed using ATIC- and ALK-specific primers (ATIC1 and ALK2 primers; Fig. 2A, lower diagram),which revealed the expected 750-bp chimeric DNA fragment (Fig.2D). In contrast, NPM and ALK primers showed no evidence for the

NPM-ALK fusion transcript (data not shown). Full-length ATIC-ALKcDNA was PCR-cloned from the patient’s RNA using specific 59-ATIC and 39-ALK primers. The ATIC-ALK fusion incorporates thefirst 229 aas of ATIC fused to the COOH-terminal 563 aas of ALK(not shown). Together, these data confirmed that RNA from thebiopsy contained a novel chimeric transcript corresponding to theATIC-ALK fusion protein.

Expression of the ATIC-ALK Fusion Protein. The full-lengthATIC-ALK cDNA was then cloned into the pCDNA3 expressionvector and transiently transfected into NIH-3T3 cells. Western blot-ting analysis using anti-ALK antibodies revealed aMr 96,000 anti-Alkimmunoreactive polypeptide (Fig. 3A,panel 2), that was not seen incells transfected with the vector alone. The difference between thepredicted ATIC-ALK MW (Mr 88,000) and that observed by Westernblotting (Mr 96,000) may be due to posttranslation modifications,including tyrosine-phosphorylation (Fig. 3A, panel 4). Side-by-sidecomparison of lysates from the patient’s lymph node and from ATIC-ALK transfected NIH-3T3 cells revealed comigration of the twoALK-proteins (Fig. 3A,panel 1).Together, these data indicate that theATIC-ALK cDNA cloned in this case corresponds to the ATIC-ALKfusion protein formed in the patient’s lymph node.

Fig. 1. Expression of ALK and NPM proteins in the patient’s lymph node biopsy.Upper, anti-Alk staining; positivity for ALK protein is restricted to the cytoplasm of thelarge anaplastic tumor cells (ALKc monoclonal antibody;31000). Lower, anti-Npmstaining; tumor cells show a nucleus-restricted expression of NPM reflecting immuno-staining of wild-type NPM (NPMa monoclonal antibody;3800). An identical stainingpattern (not shown) was observed for the COOH terminus of NPM (monoclonal antibodyNPMc). (APAAP technique; hematoxylin counterstain).

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The ATIC-ALK cDNA Encodes a Constitutively ActiveTyrosine-Kinase That Associates with Grb2 and Shc AdaptorProteins. The NPM-ALK fusion protein possesses constitutive tyro-sine kinase activity and forms stable complexes with adaptor proteins,such as Grb2 and Shc (28). Its potential to activate downstream signaltransduction pathways is demonstrated by its ability to induce consti-tutive tyrosine-phosphorylation of Shc (28). To investigate whetherATIC-ALK has a similar activation mechanism, we compared thestatus of kinase activity, the association with Grb2 and Shc, and the

ability to induce Shc tyrosine-phosphorylation of ALK-proteins inlysates from NIH-3T3 cells transiently transfected with ATIC-ALKand NPM-ALK cDNA. Anti-Alk immunoprecipitates showed compa-rable amounts of ATIC-ALK and NPM-ALK fusion proteins (Fig. 3A,panel 3) with similar content of phosphotyrosine residues (Fig. 3A,panel 4). Similarly, antiphosphotyrosine immunoprecipitates from thesame cellular lysates revealed comparable amounts of ATIC-ALK andNPM-ALK proteins (Fig. 3A,panel 6). Kinase assays of ALK-immu-noprecipitates from cellular lysates of ATIC-ALK and NPM-ALK

Fig. 2. Cloning of the chimeric ATIC-ALK cDNA.A, Schematic representation of the ALK (upper diagram) and ATIC-ALK (lower diagram) cDNAs.SS, putative ALK signalsequence;TM, ALK transmembrane domain;PTK, ALK protein tyrosine kinase domain;IMPCH, IMP cyclohydrolase domain.Arrows below the diagrams, the position and directionof the primers used;arrow above the ALK structure, the breakpoint (BP).B, agarose/EtBr gel showing the products of the 59-RACE using AP1 and ALK1 primers.1, 1-kb ladder(Boehringer Mannheim);2, diagnostic cDNA primed with AP1 primer alone;3, diagnostic cDNA primed with ALK1 primer alone;4, 59-RACE using AP1 and ALK1 primers and2.5 ng of diagnostic cDNA as template;5, 59-RACE using AP1 and ALK1 primers with 5 ng of diagnostic cDNA as template;6, 59-RACE using AP1 and ALK1 primers with 2.5ng of control cDNA as template;7, 59-RACE using AP1 and ALK1 primers with 5 ng of control cDNA as template;8, negative control of the PCR reaction.C, agarose/EtBr gel showingthe products of nested PCR.1, 1-kb ladder;2, nested PCR with AP2 and ALK2 primers using the 1.3-kb DNA fragment purified from the first round PCR, as inLane 4and6 of B;3, negative control of the PCR reaction.D, agarose/EtBr gel showing amplification of the ATIC-ALK mRNA from patient and control RNA, using ATIC1 and ALK2 primers.1, 1-kbladder;2, ATIC-ALK cDNA primed with ATIC1 and ALK2 (positive control);3, diagnostic cDNA primed with ATIC1 alone;4, diagnostic cDNA primed with ALK2 alone;5,diagnostic cDNA primed with ATIC1 and ALK2;6, control cDNA primed with ATIC1 alone;7, control cDNA primed with ALK2 alone;8, control cDNA primed with ATIC1 andALK2; 9, negative control for the PCR reaction.E, nucleotide sequence and deduced aa sequence of the 735-bp DNA fragment containing the breakpoint of the chimeric ATIC-ALKcDNA.

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NIH3T3-transfected cells showed the presence of aMr 96,000 and aMr 80,000 polypeptide respectively, representing autophosphorylatedfusion proteins (data not shown). The p52 and p46 Shc proteinscoprecipitated with both fusion proteins (though to a lesser extent inthe case of the ATIC-ALK fusion protein (Fig. 3A, panel 5).

To evaluate the association of Grb2 with the two ALK-fusionproteins, anti-Grb2 immunoprecipitates from the same lysates wereblotted with anti-Alk antibodies (Fig. 3A, panel 7). Anti-Grb2 blotsserved as controls for the efficiency of the Grb2 immunopurifica-tion (Fig. 3A, panel 8). The results showed that Grb2 coprecipi-tated with both NPM-ALK and ATIC-ALK, although again thecoprecipitation showed less Grb2-associated with ATIC-ALK than

that found in the NPM-ALK-Grb2 complex. Finally, antiphospho-tyrosine blots of anti-Shc immunoprecipitates revealed constitutivetyrosine-phosphorylation of Shc in both NPM-ALK and ATIC-ALK expressing cells (Fig. 3A, panel 10). Anti-Alk blots of thesame immunoprecipitates confirmed the existence of a stable Shc-ALK-fusion protein complex (Fig. 3A, panel 9).

It appears, therefore, that, like NPM-ALK, the ATIC-ALK fusionprotein is a constitutively active tyrosine kinase with the potential todeliver intracytoplasmic activating signals.

ATIC-ALK Expression Induces Neoplastic Transformation ofNIH-3T3 Cells. NPM-ALK is known to transform NIH-3T3 cells(28, 29), and the ATIC-ALK expression vector induced foci of trans-

Fig. 3. Expression of ATIC-ALK cDNA.A, expression of the ATIC-ALK cDNA and evaluation of its tyrosine kinase activity and effects on Grb2 and Shc.Pt, patient’s proteinlysate; all of the other lanes correspond to lysates or immunoprecipitates from NIH-3T3 cells transfected with the ATIC-ALK or NPM-ALK expression vectors or the pCDNA3 vectoralone, as indicated.Panels 1and 2, Western blots (WB) of the indicated lysates, using anti-Alk (a-Alk) monoclonal antibodies.Panels 3–10, Western blots (WB) of specificimmunoprecipitations (a-Alk, a-pY, a-Grb2, a-Shc), using the indicated antibodies.B, focus formation assay (upper panel), NIH-3T3 cells were transfected with the vector only,NPM-ALK expression vector, or ATIC-ALK expression vector, as indicated. Transformed foci were observed 10–14 days after transfection. Morphology of NPM-ALK and ATIC-ALKtransformed cells (lower panel) derived from one typical focus compared with control cells derived from vector-only transfected cells, as indicated.

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formed cells with comparable or higher efficiency (NPM-ALK andATIC-ALK yielded a comparable amount of resistant colonies inparallel dishes treated with G418 for selection of the expressionvectors; data not shown; Fig. 3B, upper panel). Cells picked from theNPM-ALK and ATIC-ALK foci showed the same morphology oftransformed fibroblasts (refractile, spindle-shaped, and elongatedcells; Fig. 3B,lower panel).

Discussion

We have reported here the cloning and expression of a novel ALKfusion protein from a case of ALK-positive ALCL. This case wasinitially suspected as genetically distinct from classic NPM-ALK-positive ALCLs when anti-Npm and anti-Alk staining of paraffinsections revealed atypical localization patterns. Indeed, anti-Alk stain-ing revealed a “cytoplasmic-only” pattern, whereas monoclonal anti-bodies directed against the NH2-terminal portion of NPM stained thenuclei of tumor cells (reflecting reactivity with wild-type NPM). Thisstudy underscores the usefulness of anti-Alk and anti-Npm antibodiesfor detecting cases containing new genetic abnormalities in ALCLs.Because the products of the genes involved in the generation of othertumor-associated fusion proteins are frequently localized to specificsubcellular compartments, screening of hemopoietic tumors with an-tibodies against the known translocation partners may lead to theidentification of other novel genetic abnormalities.

Nucleotide sequencing of the novel chimeric transcript revealed anin-frame 59-39fusion of ATIC and ALK sequences. TheATIC gene(previously named asPur H) encodes a bifunctional protein whichcatalizes the penultimate and the final steps of thede novopurinenucleotide biosynthetic pathway. It acts as a AICARFT and as anIMPCH, to catalyze the formation of FAICAR and IMP, respectively(26–27). Deletion mutant analysis of the ATIC cDNA has demon-strated that the IMPCH and the AICARFT enzymatic activities seg-regate within two nonoverlapping functional domains, with theIMPCH domain located at the NH2 terminus of the protein (30–32).The crystal structure of the ATIC protein revealed a smaller NH2-terminal globular portion (approximately from aa 1 to 200), a 30-aa-helical (linker) region, and a COOH-terminal globular region (31–32).The isolated NH2-terminal portion of the ATIC protein (aa 1–199)retains IMPCH activityin vitro, which suggests that the NH2-terminalglobular region of ATIC has independent folding properties andcontains the IMPCH enzymatic activity (31). The portion of the ATICtranscript retained within the ALK-hybrid mRNA corresponds to aa1–229 (Fig. 2E). It is, therefore, possible that the ATIC-ALK tran-script encodes a fusion protein with IMPCH activity. Thede novopurine pathway is an important target for the chemotherapeuticagents; for example, methotrexate exerts a potent inhibitory effect onAICARFT activity (33). It will be of interest to determine whether theATIC-ALK fusion protein alters purine metabolism in ALCL cellsand/or their sensitivity to purine-inhibitors.

We have not been able to investigate the chromosomal origin of theATIC-ALK recombination because of the inadequacy of patient ma-terial for cytogenetic studies. TheATIC gene has been previouslymapped between bands q34 and q35 of the long arm of chromosome2 (31), a region that is rearranged in the cryptic inversion of chromo-some 2 in cases of NPM-ALK-negative, ALK-positive ALCLs (18). Itis, therefore, possible that theATIC-ALKfusion gene is the product ofthe cryptic inv(2) (p23q35). Notably, as in our case, in all of theALCL cases with inv(2) (p23q35), the ALK protein accumulates inthe cytoplasm only (18).

The portion of ALK retained in the ATIC-ALK fusion protein is thesame as in NPM-ALK as well as in the other two variant ALK-fusionproteins thus far identified (TPM3-ALK and TFG-ALK; Refs. 20–

21). In common with NPM-ALK, ATIC-ALK possesses constitutivetyrosine kinase activity, activates cytoplasmic signal transductionpathways, and is able to transform rodent fibroblasts. The activationof the ALK kinase domain is the consequence of the constitutivehomodimerization of the fusion protein, triggered by the NPM oli-gomerization domain present in NPM-ALK (15). Likewise, TPM3andTFG contain coiled coil domains that may induce dimerization oftheir corresponding ALK-fusion proteins (34, 35). This could alsooccur with ATIC the crystal structure of which is a homodimer (31).

Fusion to an heterologous oligomerization domain is sufficient toactivate the capacity of ALK sequences to transform immortal rodentfibroblasts, which suggests that the NPM portion of the molecule (orof the other ALK-partners) is responsible only for dimerization, withno apparent further function for the delivery of mitogenic stimuli (15,36). However, the contribution of the various ALK-partners duringlymphomagenesisin vivo remains to be established. It will be ofinterest to determine whether alteration of purine metabolism contrib-utes to lymphomagenesis.

Acknowledgments

We thank Barbara Bigerna and Roberta Pacini for their skillful technicalassistance and Sara Barozzi for important experimental contributions. We alsothank Claudia Tibido and Annalisa Ariesi for excellent secretarial assistance.

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ALK-POSITIVE LYMPHOMA EXPRESSING THE ATIC-ALK FUSION PROTEIN

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2000;60:793-798. Cancer Res   Mirella Trinei, Luisa Lanfrancone, Elias Campo, et al.   Cell LymphomaProtein (ATIC-ALK) in a Case of ALK-positive Anaplastic Large A New Variant Anaplastic Lymphoma Kinase (ALK)-Fusion

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