doi:10.1182/blood-2002-05-1343Prepublished online November 7, 2002;2003 101: 1919-1927   

 Simmons, Girish Dhall, Jennifer Howes, Roberto Piva and Giorgio InghiramiRoberto Chiarle, Jerald Z. Gong, Ilaria Guasparri, Anna Pesci, Jonjing Cai, Jian Liu, William J. and plasma cell tumorsNPM-ALK transgenic mice spontaneously develop T-cell lymphomas

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NEOPLASIA

NPM-ALK transgenic mice spontaneously develop T-cell lymphomasand plasma cell tumorsRoberto Chiarle, Jerald Z. Gong, Ilaria Guasparri, Anna Pesci, Jonjing Cai, Jian Liu, William J. Simmons, Girish Dhall,Jennifer Howes, Roberto Piva, and Giorgio Inghirami

Anaplastic Large Cell Lymphomas(ALCLs) carry translocations in which theanaplastic lymphoma kinase (ALK) geneis juxtaposed to various genes, the mostcommon of which is the NPM/B23 gene.ALK fusion proteins result in the consti-tutive activation of ALK tyrosine kinase,thereby enhancing proliferation and in-creasing cell survival. A direct role forNPM-ALK in cellular transformation hasbeen shown in vitro with immortalizedcell lines and in vivo using retroviraltransfer experiments. Nonetheless, thereis no direct evidence of its oncogenicpotential in T lymphocytes, which repre-sent the most common target of ALK chi-

meras. Here, we describe a new mousemodel of lymphomagenesis in whichhuman NPM-ALK transcription was tar-geted to T cells. NPM-ALK transgenic(Tg) mice were born with the expectedmendelian distribution, normal lymphoidorgans, and a normal number and pro-portion of helper and suppressor T cells.However, after a short period of latency,all NPM-ALK Tg mice developed malig-nant lymphoproliferative disorders(mean survival, 18 weeks). NPM-ALK Tgthymic lymphomas displayed a T-cellphenotype characteristic of immaturethymocytes and frequently coexpressedsurface CD30. A subset of the NPM-ALK

Tg mice also developed clonal B-cellplasma cell neoplasms. These tumorsarose in peripheral lymphoid organs(plasmacytomas) or within the bonemarrow and often led to peripheral neu-ropathies and limb paralysis. OurNPM-ALK Tg mice are a suitable modelto dissect the molecular mechanismsof ALK-mediated transformation and toinvestigate the efficacy of new thera-peutic approaches for the treatmentof human ALCL in vivo. (Blood. 2003;101:1919-1927)

© 2003 by The American Society of Hematology

Introduction

Human Anaplastic Large Cell Lymphomas (ALCLs) are a uniquesubset of lymphomas partly distinguished by their coexpression ofthe CD30 antigen.1 Classical cytogenetic studies demonstrated thatALCLs carry unique translocations within the p23 region ofchromosome 2.2-4 In 1994, Morris et al5 cloned the t(2;5) transloca-tion and discovered that a novel tyrosine kinase gene, the anaplasticlymphoma kinase (ALK), was fused to the NPM/B23 gene. NPMparticipates in nucleocytoplasmic trafficking6,7 and has been re-cently shown to regulate the duplication of centrosomes.8 The ALKgene encodes a tyrosine kinase receptor whose physiologic expres-sion is largely limited to neuronal cells.9,10 However, the physi-ologic role of the ALK receptor remains largely unknown becauseALK�/� mice appear normal.11 Nonetheless, ALK is phylogeneti-cally highly conserved,9,10 suggesting that it might have animportant role in neuronal cellular function. In fact when constitu-tively activated in the rat pheocromocytoma cells PC12, ALK leadsto neuronal differentiation and provides antiapoptotic signals instress conditions12 (data not shown). Recently, Stoica et al13 havealso demonstrated that pheotrophin binds to ALK receptor,3 butother additional ligands might exist.

In the past 5 years, several groups have successfully cloned newALCL translocations and demonstrated that the ALK gene can fuse

to multiple targets, which include the TFG, TPM3, ATIC, CLTCL,RanBP2, and MSN genes.11 Proteins fused to ALK largely deter-mine the subcellular localization of the derived fusion proteins,being cytoplasmic (ATIC-, TGF-ALK, etc), cytoplasmic andnuclear (NPM-ALK), or membranous (MSN-ALK).11 Moreover,ALK translocations can also be detected in nonlymphoid neoplasmssuch as inflammatory myofibroblastic tumors,14 and ALK expres-sion has been described in neuroblastomas15 as well as in a uniquesubtype of immunoglobulin A (IgA)–positive plasmacytoid tumors.16

Cellular transformation by NPM-ALK has been demonstratedin immortalized rodent fibroblasts17 and confirmed in studies thathave shown that ALK protects Ba/F3 and PC12 cells frominterleukin-3 or growth factor withdrawal13,17 (data not shown).Transfer of NPM-ALK–transduced bone marrow cells into irradi-ated host recipient mice resulted in the generation in vivo of largecell B-cell lymphomas.18 In the past few years, the molecularmechanisms of NPM-ALK–mediated cellular transformation havealso been partially elucidated.11 It has been shown that the ALKportion of the fusion protein, corresponding to the cytoplasmic tailof the ALK receptor and containing the catalytic domain, isabsolutely required for transformation,17 whereas all the N-terminal regions of the ALK chimeras function as dimerization

From the Department of Pathology and Kaplan Comprehensive Cancer Center,and Department of Pediatric Oncology, New York University School ofMedicine, New York; Department of Pathology and Centro di Ricerca inMedicina Sperimentale (CERMS), University of Torino, Torino, Italy;Department of Pathology, University of Verona, Verona, Italy; Department ofPathology, University of Cornell, New York, NY.

Submitted May 30, 2002; accepted August 21, 2002. Prepublished online asBlood First Edition Paper, November 7, 2002; DOI 10.1182/blood-2002-05-1343.

Supported by grant RO1-CA64033 from the National Institutes of Health and by

an Associazione Italiana per la Ricerca sul Cancro (AIRC) grant.

Reprints: Giorgio Inghirami, New York University, Department of Pathologyand Kaplan Cancer Center, 550 First Ave, New York, NY 10016; e-mail:inghig01@med.nyu.edu.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2003 by The American Society of Hematology

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domains.11,19 As a result of spontaneous dimerization, ALK under-goes autophosphorylation and becomes catalytically active. Consti-tutively active ALK fusion proteins can bind multiple adaptorproteins and activate a series of pathways involved in cellproliferation, transformation, and survival. These include thephospholipase c� (PLC-�) phosphatidylinositol 3 kinase (PI3K)/Akt and the Janus kinase 3/signal transducer and activator oftranscription 3 (Jak3-Stat3) pathways.17,20-22 All these moleculesand their putative roles were identified by using either nonhemato-poietic cells or immortalized B cells, leaving the molecularmechanisms of T-cell transformation by ALK chimeras stillunknown. Moreover, the recent discovery of healthy individualscarrying lymphocytes with NPM-ALK or ATIC-ALK transloca-tions23 suggests that ALK-mediated T-cell transformation is acomplex event that requires multiple and still unknown steps.

To unveil the role of ALK in T-cell transformation, wegenerated a mouse model in which expression of NPM-ALK wastargeted to T lymphocytes. All NPM-ALK transgenic (Tg) micedeveloped clonal lymphoproliferative disorders after a short periodof latency. In addition to T-cell lymphomas, a sizable fraction ofour mice also acquired plasma cell neoplasms. Studies using theseNPM-ALK Tg mice will allow a better understanding of themolecular mechanisms and genetic defects leading to ALK-mediated transformation.

Materials and methods

NPM-ALK Tg mice, cell lines, and statistical analysis

NPM-ALK transgenic mice were generated by injecting Swiss-Websterblastocysts with a construct in which the full-length cDNA of NPM-ALKchimera was placed under the control of the murine CD4 promoter. Thetransgenic cassette (CD4 cassette) included the minimal CD4 enhancer(339 base pair [bp]), the minimal murine CD4 promoter (487 bp), thetranscription initiation site, and 70 bp of the untranslated first exon and partof the first intron of the murine CD4 gene but lacked the CD8 silencer.24 TheNPM-ALK founders were back-crossed into Balb/c and C57B/6 strains andhoused in a germ-free facility (Skirball Institute of Biomolecular Medicine,New York University School of Medicine, NY).

Positive NPM-ALK mice were detected by polymerase chain reaction(PCR) by using genomic DNA obtained from mouse tail biopsies aspreviously described.25 All experiments presented in this study werederived from mice (C57BL/6 and Balb/c backgrounds) obtained from 2independent transgenic lines (N1 and N16). �cul1 Tg mice were obtainedby placing (SacI/SalI) the human Cul1-N252 cDNA (encoding 1 to 252[N252] amino terminal residues) into the CD4 cassette Tg.26 Screening offounder animals and their corresponding offspring was performed by PCRand confirmed by Southern hybridization on genomic DNA from tailbiopsies. Rag2�/� mice were purchased from Taconic (Germantown, NY).

Primary NPM-ALK cells were obtained from fresh thymic tumors afterbeing cultured in complete RPMI-1640 medium in vitro.

Survival curves were performed by using the nonparametric model ofKaplan-Meier.

Immunoprecipitation and Western blot analysis

Tissue samples and cell lines were lysed (50 mM Tris-HCl (tris(hydroxy-methyl)aminomethane) pH7.4, 150 mM NaCl, 0.1% Triton X-100, 5 mMEDTA (ethylenediaminetetraacetic acid), 1 mM Na3VO4, and 1 mMphenylmethylsulfonyl fluoride (PMSF), and protease inhibitors), and super-natants were then used for immunoprecipitation and Western blottinganalysis. For immunoprecipitations, 0.2 to 0.5 mg total proteins wereincubated for 1 hour at 4°C with 3 �g rabbit anti-ALK antibody (Ab) or acocktail of mouse anti-ALK monoclonal antibodies (Mabs; Zymed, SouthSan Francisco, CA), or with anti–Grb-2 Ab (SC255; Santa Cruz Biotechnol-

ogy, Santa Cruz, CA), anti-PI3K Ab (UBI Biotechnology, Waltham, MA);then 30 �L protein G–Sepharose beads (1:1) were added for 30 minutes.Immunocomplexes were washed (3 times with the lysis buffer) andsubsequently loaded onto a sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE) gel. For Western blotting, 30 �g proteins wererun in SDS-PAGE gels and transferred onto nitrocellulose membranes.Membranes were blocked (5% low-fat milk in phosphate-buffered saline[PBS] with 0.1% Tween 20 for 1 hour at room temperature [RT]) andsubsequently incubated with the primary antibodies for 1 hour at RT (rabbitanti-ALK [1:4000; Zymed]; mouse anti-ALK Mab [1:5000; Zymed];antiphosphotyrosine [PY20; 1:1000; Transduction Lab-Becton Dickinson,Mountain View, CA]; anti–Stat3 Mab [1:1000, Zymed]; anti–Jak-1, Jak2,Jak3, Tyk-2 Ab [1:500; Zymed]; anti-Shc Ab [1:500; Santa Cruz]). Filterswere washed 3 times and then incubated with horseradish peroxidase(HRPO)–conjugated goat antimouse or antirabbit (1:2000; Amersham,Arlington Heights, IL, for 1 hour at RT) antibodies. Immunocomplexeswere detected by using a chemiluminescence system (ECL; Amersham,Piscataway, NJ).27

Southern blotting

Southern blotting was performed as previously described.25 Briefly, high-molecular-weight genomic DNAs (10 �g) were digested by EcoRI, HindIII,or Pvu endonucleases, and then digested fragments were separated byelectrophoresis. DNAs were subsequently transferred onto nitrocellulose.Radiolabeled cDNA probes were used to study the genomic configurationof T-cell receptor � (TCR�) and heavy-chain immunoglobulin loci.28

Human NPM-ALK genomic sequences were investigated using BamHI-digested DNAs using a specific ALK cDNA probed (BamHI-BamHI).

Flow cytometry, histology, and immunohistochemistry

Single-cell suspensions were obtained from isolated tissue samples. Cellswere washed, counted, and stained with the following murine primaryfluorescein isothiocyanate (FITC)–, phycoerythrin (PE)–, or Tricolor-conjugated antibodies: Thy-1, CD4, CD8, B220, CD25, CD3, TCR �/�,TCR �/� (Caltag Laboratories, Burlingame, CA), CD30, CD44, andCD45RB (Pharmingen-BD Biosciences, San Jose, CA). After staining (30minutes at 4°C), cells were washed and analyzed by using a fluorescence-activated cell sorter scan (FACScan; Becton Dickinson) flow cytometeras described.25

For the histologic and immunohistochemistry analyses, tissue sampleswere fixed in PBS-buffered formalin (10%) and subsequently embedded inparaffin. Dewaxed 4-�m–thick tissue sections were stained with hematoxy-lin and eosin or after microwave retrieval (citrate buffer, pH 6.6, 15minutes) incubated with anti-ALK primary antibody (1:1000; Zymed),anti–Ki-67 (Novacastra), anti-CD45R (B220; 1:100; Caltag), and anti-CD138 (1:20; Pharmingen-BD). Bound complexes were revealed by usingthe avidin biotin peroxidase complex and a semiautomated immunostainer(DAKO, Carpinteria, CA; or Ventana ES Medical Systems, Tucson, AZ).Mouse light- and heavy-chain expression was performed by using alkaline-conjugated rabbit antimouse antibodies (Southern Biotechnology Associ-ates, Birmingham, AL). For immunofluorescence stains, paraffin-embeddedtissue sections were treated as described earlier. Sections were thenincubated with rabbit anti-ALK Ab. After washing, tissue sections wereincubated with biotin-conjugated antirabbit Ab (1:200; Vector) and thenFITC-Avidin (1:200; Sigma-Aldrich, St Louis, MO). Sections were subse-quently incubated with normal rabbit serum (1:10, 30 minutes at RT) andthen stained with PE-conjugated anti-B220 (1:20; Caltag) in presence ofrabbit serum (1:10). After washing, slides were briefly dried and cover-slipped with antifade (Vysis, Downers Grove, IL). Fluorescence stainingwas visualizes by using the 2.7 Cytovision software (Applied Imaging,Santa Clara, CA).

Tissue culture

The rate of spontaneous and in vitro–induced cell death was evaluatedaccording to DNA content and propidium iodide or Annexin V (Pharmingen-BD) stainings.25 Briefly, thymocytes were cultivated with immobilized

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anti-CD3 antibody (10 �g/mL; 2C11; a gift of J. Bluestone) and solubleanti-CD28 antibody (5 �g/mL; Pharmingen-BD). Alternatively thymocyteswere cocultured with dexamethasone (0.1 �M), tumor necrosis factor(TNF; 15 ng/mL), cycloheximide (75 �g/mL; Sigma-Aldrich), anti-Fas Ab(0.5 �g/mL; Pharmingen-BD), phorbol 12-myristate 13-acetate (PMA; 10ng/mL; Sigma-Aldrich), or Ionomycin (1 �M; Sigma-Aldrich). At theindicated times, cells were harvested, washed, and stained.

Purified peripheral T cells were obtained by magnetic-bead separation.Briefly, 1 � 107 lymph node cells were first incubated (for 30 minutes atRT) with a cocktail of antibodies (0.2-0.5 �g each antibody/106 cells)against B cells (B220; Caltag), macrophages (CD11c; Caltag), and naturalkiller (NK) cells (Anti-NK; Caltag). At the end of incubation, 70 �Lantirat-conjugated magnetic beads (Dynabeads; Dynal, Lake Success, NY)were added. Bead-coated cells were separated in a magnetic field, andunbound cells were washed in cold PBS (3 times). Negatively selected Tcells were first stained with FITC-conjugated anti-CD3 mAb and analyzedby FACS to determine their purity (always 95%). Highly purified T cells(5 � 104) were cultivated in RPMI-1640 medium supplemented with 10%fetal calf serum (FCS), streptomycin and penicillin, and 10�5 M 2-mercap-toethanol in 96-well plate coated with anti-CD3 (0.1-10 �g/mL) andsoluble anti-CD28 (0.5 �g/mL) antibody for 48 hours. Alternativelypurified T cells were cocultured with PMA (10 ng/mL) or concanavalin A(ConA; 5 �g/mL). 3H-thymidine (0.037 MBq/well [1 �Ci/well]; NewEngland Nuclear, Boston, MA) was added for the last 18 hours of culture.Cells were harvested and counted in a 5000TD scintillation counter(Beckman, Fullerton, CA).

Electrophoretic methods

Semiatutomated agarose electrophoresis and immunofixation were per-formed on HYDRASYS and HYRYS systems (Sebia, Norcross, GA)according to the manufacturer’s instructions. For protein electrophoresis,10 �L sample was applied manually to the sample template. The subsequentsample application, electrophoresis (pH 8.6, 20 W, at 20°C), gel drying, andstaining were performed automatically. The resulting electrophoretic pro-files were scanned using the Hyris densitometer (Sebia). For immunofix-ation, each sample was applied in 6 different positions on agarose gels(Hydragel Immunofixation; Sebia), and the electrophoretic separationperformed automatically under identical conditions as earlier. Eitherfixative or monospecific antisera to mouse immunoglobulins (, �, IgG,IgM, and IgA; Southern Biotechnology Associates) were applied to theelectrophoresis lanes to allow for fixation and immunoprecipitation,respectively. Detection of monoclonal bands was assessed by visualinspection of stained gels.

Results

NPM-ALK is expressed in normal T cells

To study the influence of NPM-ALK in T cells of mice, thefull-length cDNA of NPM-ALK fusion gene was cloned in vectorunder the control of the murine CD4 promoter (Figure 1A).Injection of this construct into blastocysts yielded 6 differentNPM-ALK founders that were identified from 3 foster mothers.The copy number of the NPM-ALK transgene varied considerablyamong the different lines (Figure 1B). With the exception of onemouse (N8), all founders and their corresponding NPM-ALKprogenies (N1, N14, N16) expressed the expected ALK fusionprotein with a molecular weight of 80 kDa (Figure 1C). Thisprotein corresponded to the NPM-ALK of human cell linescarrying the t(2;5) translocation and was expressed at levels similarto those of human ALCL-derived cell lines (Figure 1C). All 5NPM-ALK–expressing founders were crossed to generate 5 differ-ent mouse lines. However, N5 and N15 died, before mating, ofbilateral posterior limb paralysis and thymic tumor, respectively.

The CD4 transgene cassette allows the expression of the targetprotein in all T cells, including early progenitor thymocytes(CD4�/CD8�) and single positive T cells (CD4�/CD8� andCD4�/CD8�).25 As predicted, the transgenic NPM-ALK proteinwas localized to cortical and medullary thymocytes, lymphocyteswithin the interfollicular areas of lymph nodes, and in the T-cellareas of the splenic white pulp (Figure 1D). NPM-ALK wasdetected in the cytoplasm and nucleus, a pattern similar to thatobserved in human NPM-ALK� cells.

Stat3 and Jak3 are constitutively phosphorylated in NPM-ALKTg mice

Because NPM-ALK is constitutively autophosphorylated in humanALCL cells, we analyzed the phosphorylation status of NPM-ALKin transgenic cells and observed that in normal, as well asneoplastic NPM-ALK cells, it is constitutively phosphorylated(Figure 2A). Because activated ALK fusion proteins can efficientlybind Shc, PLC-�, Grb-2, and PI3K,29 we studied whether thetransgenic NPM-ALK fusion protein could efficiently bind the

Figure 1. Generation of NPM-ALK Tg mice. (A) NPM-ALK cDNA was cloned into aconstruct containing the CD4 enhancer and promoter as described in “Materials andmethods.” (B) Southern blotting of representative animals obtained from differentfoster mothers. BamHI-digested DNAs were hybridized with a radio-labeled ALKcDNA probe (1 N1, 2 N16, 4 N15, and 8 N8. Lane 3, 5, 6, and 7 correspondto correspondent normal littermate). (C) The expression and size of the fusion proteinwas characterized by Western blot. Proteins were extracted from thymi of Tg (N1,N14, and N16) and wild-type (WT) mice and loaded onto SDS-PAGE gel. Theexpression of the NPM-ALK chimera was detected with polyclonal rabbit anti-ALKantibody. The protein extract from the human ALCL-derived cell line DHL was used asa control. The loading was checked by Western blot for the ubiquitous CDK2 protein.(D) Histology of NPM-ALK Tg mice. Tg thymus (left panels) or spleen (right panels)were fixed in formalin and embedded in paraffin. Hematoxylin and eosin stains (toppanels, � 100) showed normal thymus and spleen architecture in the preneoplastictissue. Immunostaining with anti-ALK antibody (bottom panels) demonstrated adiffuse positivity in Tg thymocytes with stronger signal in medullary lymphocytes(� 100). In Tg spleen the ALK positivity was localized in the periarteriolar T cell areasof the white pulp (� 400). Left insert shows a nuclear and cytoplasmic staining in Tglymphocytes (� 400); right insert shows a lower magnification of the spleen (� 100).

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corresponding mouse proteins as well. As shown in Figure 2B,mouse Shc, IRS-1, Grb-2, and PI3K proteins efficiently boundNPM-ALK in normal as well as in neoplastic cells. Moreover, wewere able to demonstrate that phosphorylated Stat3 could becoprecipitated with ALK (data not shown). Because NPM-ALKleads to the constitutive activation of Stat3,21,22 and Jak3,22 wefurther investigated the activation status of these molecules in ourNPM-ALK Tg mice. As shown in Figure 2C-D, NPM-ALK Tgthymocytes, but not control cells, displayed constitutively phosphor-ylated Stat3 and Jak3. Overall, these findings demonstrate that theNPM-ALK transgene is constitutively activated in T cells andbinds to the same adaptor proteins as in humans. Thus, ourtransgenic model mimics the molecular features of humanNPM-ALK� lymphomas.

Cellular phenotype and lymphoid organ development inNPM-ALK transgenic mice

To characterize the putative effects resulting from the constitutiveactivation of NPM-ALK in T lymphocytes, we analyzed themorphologic and phenotypic features of T-cell lymphoid popula-tions and their activation and differentiation states. Overall, therelative and absolute numbers of T and B lymphocytes, withinprimary and secondary lymphoid organs, were similar in Tg andcontrol littermate mice. Microscopic evaluation demonstrated a

normal lymphoid organization with the physiologic preservation ofall lymphoid microenvironments. Finally, the histologic surveys oflung, kidney, stomach, intestine, testis, ovaries, and brain did notreveal any morphologic anomalies.

Flow cytometry of NPM-ALK Tg thymocytes showed a normaldistribution of CD4�/CD8� and CD4�/CD8� cells as well as ofsingle positive CD4� or CD8� lymphocytes (Figure 3A). Nosignificant differences were observed in the expression of other Tcell-associated and/or -restricted markers. A normal percentage andexpression of V� chains and/or the CD3 complexes were alsodocumented in transgenic T lymphocytes, demonstrating that T-cellcommitment and maturation proceed normally in these mice (datanot shown). The peripheral lymphoid organs showed a normalproportion of B and T lymphocytes and a normal ratio of the CD4�

and CD8� populations (Figure 3A). Finally, the percentage ofactivated peripheral T lymphocytes was similar in Tg and WT miceas demonstrated by expression of CD25 and CD69 antigens(3%-5% of the total cells). Flow cytometry of spleen showed nosignificant differences in the distribution of myeloid, erythroid, orgranulocytic lineages. Double immunofluorescence studies werealso performed to address whether mature T and/or B cells couldexpress NPM-ALK. As shown in Figure 3B, NPM-ALK expres-sion (nuclear green staining) was restricted to B220/CD45R� cells(B220/CD45R� cells showed only a red membrane staining)present in the T-cell areas of splenic germinal centers, suggestingthat NPM-ALK was largely restricted to T lymphocytes.

To determine whether the constitutive expression of NPM-ALKcould possibly modify the survival and/or proliferative potential ofT lymphocytes, NPM-ALK Tg thymocytes were incubated in vitrowith different apoptotic stimuli. As shown in Figure 3C, both Tgand controls had similar rates of spontaneous and induced apopto-sis. The in vitro proliferative rates of purified peripheral Tlymphocytes, stimulated with suboptimal and to “ad hoc” concen-trations of mitogens, were also similar in transgenic and controlmice (Figure 3D). These findings indicate that NPM-ALK alone isnot capable of significantly modifying the survival and cell growthof T lymphocytes from young mice in vitro.

NPM-ALK transgenic mice develop spontaneouslymphoid tumors

Mice from N1, N14, and N16 lines were healthy up to 5 to 7 weeksof life. After the fifth week, Tg animals started to develop tumors.Survival curves obtained from 86 mice for the N16 line and 110mice for the N1 line showed a mean survival of 18.5 (Figure 4A),and 17 weeks (Figure 4B), respectively, with a overall incidence of100% for both lines. Tumors were mainly represented by thymiclymphomas or plasma cell neoplasms (Figures 5 and 6), and all 3lines developed, albeit with different frequencies, both thymiclymphomas and/or plasma cell tumors. Mice belonging to the N1line showed, in fact, a prevalence of plasma cell tumors ( 80%),in contrast to N16 mice that more often developed thymiclymphomas ( 90%). Thymic and plasma cell tumors occurredwith a similar frequency (50%) in mice of the N14 line. In rarecases (� 5% overall), we also found neoplasms characterized byatypical, spindle cells within a dense connective tissue. In addition,we also documented rare tumors (� 1%) characterized by imma-ture cells with abundant cytoplasm lacking either T- or B-cellmarkers, but expressing CD11b. These tumors involved central andperipheral lymphoid tissues and were often observed infiltrating theliver, kidneys, lungs, and other internal organs.

The mediastinal T-cell lymphomas were composed of medium-sized lymphoblasts, with a relatively high mitotic index (10-15

Figure 2. Molecular characterization of NPM-ALK Tg mice. (A) Expression andconstitutive activation of NPM-AK in Tg mice. Thymocytes from Tg and WT mice werelysed and immunoprecipitated with anti-ALK Ab as described in “Materials andmethods.” Western blot with anti-ALK revealed the presence of the protein in Tg butnot WT mice (left panel). NPM-ALK protein was constitutively phosphorylated in Tgmice as revealed by the antiphosphotyrosine Ab (right panel). (B) NPM-ALK proteinexpressed in murine T cells coprecipitates with Shc, IRS-1, Grb-2, and PI3K. Lysedfrom ALK� samples were immunoprecipitated with anti-ALK (upper panel) or withanti–Grb-2 or anti-PI3K (lower panel). Immunocomplexes were gel electrophoresedand, after transfer, incubated with the indicated antibodies. Direct Western blottingwere also performed as indicated. All data are representative of at least 3 differentexperiments. (C) NPM-ALK Tg mice activate Stat3. Proteins extracted from Tg andWT thymocytes were immunoprecipitated with anti-Stat3 Ab and loaded onto aSDS-PAGE gel. Stat3 protein was similarly immunoprecipitated from both Tg and WTthymocytes. Antiphosphotyrosine Ab revealed the presence of higher levels ofactivated Stat3 in NPM-ALK Tg mice. (D) NPM-ALK Tg mice activate Jak3. Jak-familyof proteins were immunoprecipitated from Tg and WT mice and detected with antiphospho-tyrosine Ab. Only Jak3 was constitutively phosphorylated in Tg but not in WT mice. TheJak-family of proteins were equally immunoprecipitated in Tg and WT mice.

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mitosis/10 high power field [hpf]) and high proliferation index asdemonstrated by anti–Ki-67 staining (Figure 4C). These immaturethymocytes were always Thy-1� and CD44� but B220�. Theexpression of other antigens was variable. Most of the tumorslacked CD4, CD8, CD3, and TCR, but a fraction was CD4�/�/CD8�, CD3�/�, and CD30� (Figure 4D). Their clonal nature wasdocumented by Southern blot analysis (Figure 4E). Moreover, in alimited number of cases we performed classical cytogeneticanalysis, which documented a normal karyotype (data not shown).Finally from a representative group of these tumors, we established9 different cell lines, whose immunophenotypes matched those ofthe corresponding primary tumors. All these tumor cell lines grewefficiently in soft agar and in immunodeficient mice (Rag2�/�)after subcutis or intravenous injections (Figure 4F).

Plasma cell tumors could be categorized into 3 major groups,based on their cytologic features. The first group included tumorscomposed primarily of mature plasma cells characterized by a largecytoplasm and eccentric and sometime binucleated nuclei withevident nucleoli. The second group included tumors with large,atypical cells with irregular nuclei and conscious nucleoli. Finally,a subset of these neoplasms displayed very atypical, pleomorphic/anaplastic cells (Figure 5A-D). Plasmacytomas occurring in lymphnodes, spleen, and very rarely the thymus often completelyreplaced these lymphoid organs and invariably invaded the surround-ing tissues. Furthermore, in a substantial subset of the transgenicmice (20%), the neoplastic plasma cells occupied the bone marrowspaces and invaded into the vertebral bones, compressing and often

destroying spinal ganglia and nerves (Figure 6A,B). In rareinstances the neoplastic cells, growing within the perispinal spaces,even reached the central nervous system (Figure 6C). Thesehistologic findings corroborated the frequent gross limb paralysisof these mice and other postural and behavioral (spinning androtational) habits. Notably, these plasma cell tumors occurred withthe same frequency in mice crossed in C57BL/6 and Balb/cbackgrounds. Immunophenotypic analysis of these neoplasmsdemonstrated that these tumors rarely expressed B220/CD45R butwere invariably NPM-ALK� (Figure 6D) and CD138� (Figure6E). The proliferation rate as measured by the Ki-67 staining wasvariable ranging from 10% to 40% (Figure 6F). The B-cell origin ofthese tumors was further confirmed by the Southern blotting(Figure 7A) and by enzyme-linked immunosorbent assay (ELISA;data not shown). Furthermore, immunohistochemical stainingperformed on paraffin-embedded tissue samples demonstrated theclonotypic expression of heavy and light chain of these tumors(Figure 6G). Moreover, free light chain immunoglobulin wasdemonstrated in animals carrying plasma cell neoplasms (Figure6H-I). Collectively, these findings demonstrate that these neo-plasms express clonal immunoglobulin, which can be secreted anddetected in the serum.

Finally, we investigated the expression profiles of several cellcycle regulators and Stat3 and Stat5 in fresh tumor samples and in 3NPM-ALK T-cell lines. All NPM-ALK� samples showed theconstitutive expression of phosphorylated Stat3 (Figure 7B).However, a single NPM-ALK case displayed very low levels of

Figure 3. Normal phenotype of NPM-ALK Tg mice. (A)Single cell suspensions obtained from thymocytes, spleen,and lymph nodes were stained with the indicated antibodiesand analyzed as described in “Materials and methods.” Tg andWT mice had comparable phenotype in both immature andmature T cells and B lymphocytes. (B) NPM-ALK expression inmature T and B lymphocytes: NPM-ALK expression is re-stricted only to T cells. Paraffin-embedded tissue section froma preoplastic of NPM-ALK Tg spleen mouse was stained withanti-ALK (green) and anti-B220 (red) Abs. Normal response ofTg lymphocytes to apoptotic and proliferative stimuli. (C) Tgand WT thymocytes were isolated and stimulated for 24 hourswith the indicated reagents. The spontaneous and inducedapoptotic rate was comparable in Tg and WT mice. (D)Peripheral T lymphocytes were purified from lymph nodes asdescribed in “Materials and methods” and cultured for 72hours in the presence of the indicated reagents. 3H-thymidinewas added for the last 18 hours of culture. Proliferativeresponses of WT and Tg mice were comparable. The data arerepresentative of at least 2 independent experiments.

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phosphorylated Stat5, despite the relatively high levels of Stat5.Interestingly the expressions of c-myc, phospho-Erk-1/2, andcyclin A and D3 were similar in NPM-ALK and in �cul1 tumors(Figure 7B). We decided to use �cul1 tumors as control because

Figure 4. NPM-ALK Tg mice develop lymphomas. Survival curves NPM-ALK Tglines N16 (A) and N1(B). (C) Thymic lymphomas. Thymic lymphomas were com-posed of a homogeneous population of medium-sized lymphoid cells. Numerousmitosis and apoptotic bodies were present (left panel, � 100) (Ki67� cells weredocumented by immunohistochemistry, insert; � 200). Immunohistochemical stain-ing with anti-ALK Ab demonstrated a nuclear and cytoplasmic expression of theNPM-ALK fusion protein (right panel, � 100). (D) Typical phenotype of thymiclymphomas. Tumor cells obtained from neoplastic thymus were stained with theindicated Abs and analyzed (Thy1�, B220�, CD44�, CD8�, CD4�/�, CD25�). (E)Southern blot analysis of NPM-ALK lymphomas showing a rearranged pattern of theT-cell receptor with all the enzymes used for digestion. Germline liver DNA was usedas control. (F) NPM-ALK T-cell lines established tumors in immunodeficient mice.Tumor cells (2 � 106) (NPM-ALK-Ova) were injected subcutis, and animals werefollowed daily for 4 weeks (upper panel). Tumors were composed of medium-sizedblasts (lower panel, � 400) with high proliferation index (anti-Ki67 staining in theinsert, � 200).

Figure 5. NPM-ALK Tg mice develop plasma cell tumors. (A-D) Histologicsections of 4 representative plasma cell neoplasms (�400).

Figure 6. Plasma cell immunophenotype and clonality. Plasma cell involving thebone marrow replaced the normal bone marrow and disrupted the bone trabeculae(A, � 200). Neoplastic plasma cells often infiltrated the perispinal tissues andganglions (B, � 200) and in some cases invaded the central nervous system(C, � 200). NPM-ALK was largely confined within the cytoplasm of the neoplasticplasma cells (D, � 400). Tumor cells were invariably CD138� (E, � 400), displayed avariable number of Ki-67� cells (F, � 400) and they expressed clonotype heavy andlight immunoglobulin determinants (G, � 100). The serum analysis also demon-strated the presence of free light chain immunoglobulin (H-I).

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these tumors show a phenotype similar to that observed inNPM-ALK mice and because the �cul1 expression was achievedusing the same transgenic cassette. Overall, these findings confirmthat NPM-ALK Tg neoplastic T cells express high levels ofphosphorylated Stat3 and parallel the findings in human ALCL.21,22

Discussion

We have produced and characterized a new mouse model ofNPM-ALK–induced lymphomagenesis and have demonstrated thathuman NPM-ALK leads invariably to the generation of T-celllymphomas and plasma cell tumors. Our findings show that ALKcan efficiently bind a series of mouse adaptor proteins and result inthe constitutive activation of Jak3 and Stat3. Nonetheless, innormal cells NPM-ALK alone does not increase cell proliferationand/or promote the survival of normal thymocytes toproapototic agents.

Even though several groups have demonstrated the transform-ing potential of ALK chimeric proteins in vitro, and Kuefer et al18

have shown that NPM-ALK–containing retrovirus can lead to thetransformation of hematopoietic cells toward B-cell large celllymphomas, the ability of NPM-ALK to induce the transformationof T lymphocytes in vivo is still under debate. Indeed, understand-ing the pathogenetic role of NPM-ALK in T-cell transformation isimportant because most of the ALK� human ALCLs are T cell inorigin30 and ALK� lymphocytes have been detected in healthyindividuals.23

Our in vivo studies demonstrated that the constitutive activationof ALK can successfully prompt, with a relatively short latency,spontaneous lymphomagenesis in all mice. This is particularlyinteresting considering that in other murine transgenic modelsT-cell tumors occur in only a subset of the animals.28,31,32 Theefficient ability of activated ALK to induce transformation may bedue to the diversity and complexity of the ALK-signaling pathway.In fact, we and others have shown that PI3K, PLC-�, Ras, andJak3-Stat3 pathways can be simultaneously activated.17,21,22,29

Interestingly, these pathways not only enhance cell growth but alsoprovide antiapoptotic signals, in some cases by synergisticallyaugmenting their individual effects. Toward this end, the activationof Stat3 and PI3K provide synergistic survival signals via Bcl-xL22

and AKT,29 respectively. Growth signals can be also achieved viaStat3 and its downstream effectors, mainly cyclinD1 and c-myc(data not shown), and Ras via Erk1/2.

In our model the T-cell tumors were exclusively lymphoblasticlymphomas. The precise mechanism(s) for this prevalence isunclear. Because thymocytes proliferate rapidly, it is possible thatcell proliferation may be an important requirement for the ALK-mediated transformation observed in our model. A relatively highrate of cell division may allow the acquisition of a sufficientnumber of genetic alterations capable of cooperating with ALK,thereby spurring transformation. Alternatively, the transformationof NPM-ALK thymocytes may occur because immature T cellsexpress unique genes, which may promote the activation of uniquepathways or facilitate genetic aberrations. For example, immaturethymocytes actively undergo gene rearrangement of their TCR loci,and they are likely to undergo erroneous genetic recombination.33

These errors might lead to cell death but in the presence of ALKmay fortuitously encourage or promote transformation.

Immunophenotypic analysis showed that a fraction (approxi-mately 50%) of ALK T cells coexpressed the CD30 antigen, amolecule that is invariably expressed by human ALCL.1 Because avery small subpopulation of normal thymocytes express detectablesurface CD30,34 it is unclear whether the NPM-ALK CD30�

tumors may simply derive from these normal CD30� thymocytesor whether CD30 could be up-regulated as a result of ALKexpression or cell transformation. If NPM-ALK directly regulatedthe expression of CD30, one could predict the overexpression ofCD30 in normal NPM-ALK Tg thymocytes. However, the analysisof normal transgenic cells did not show any CD30 up-regulation.Thus, transformed NPM-ALK� cells may undergo nonspecificchanges, such as chromatin remodeling, etc, which facilitate thetranscription of other genes, including CD30, via transcriptionfactors whose expression might be ALK mediated. These hypoth-eses require additional studies.

Together with T-cell lymphomas, NPM-ALK Tg mice alsodeveloped ALK� plasma cell tumors. The B-cell origin of thesetumors was confirmed by the presence of specific heavy-chainimmunoglobulin gene rearrangements and by expression of B-cell/plasma cell–associated antigens CD45R and CD138. Our resultsexclude the possibility that ALK tumors may develop as a result ofthe unique insertion of the transgene in proximity of genesexpressed in B cells and/or responsible for the activation of targetgenes that facilitate B-cell transformation, because all the 3transgenic lines developed such tumors. The constitutive expres-sion of ALK in these neoplastic plasma cells in addition to theirhigh incidence in NPM-ALK Tg mice strongly suggest that theoccurrence of plasma cell neoplasms is due to the forced expressionof ALK. Nevertheless, our CD4 cassette should allow the expres-sion of the desired transgene only in T cells.24 The aberrantexpression of ALK in the neoplastic plasma cells suggests that thiscassette may be transcriptionally active in some B cells that may becommitted to plasma cell differentiation and/or or in plasma cells.The expression of CD4 in normal B cells has not been described.However, the absence of the CD8 silencer in our construct mayresult in the inappropriate expression of the driven transgene. Thelow level of expression of CD30 in some non–T cells in CD30 Tgmice tends to support this hypothesis (data not shown). Alterna-tively, plasma cells may aberrantly transcribe CD4 and thus mighthave the appropriate transcription machinery to induce the expres-sion of NPM-ALK in our transgenic B cells.35-37 Finally, Delsol etal16 have described a group of IgA� plasmacytoid B cell tumorsthat overexpresses ALK and CD4 antigens.16

Regardless of the precise mechanisms leading to the aberrantexpression of ALK in Tg plasma cells, NPM-ALK Tg mice are asuitable model to study plasma cell tumors and, in particular,

Figure 7. Plasma cell and NPM-ALK tumors. (A) Southern blot analysis of plasmacell tumors showing rearranged pattern of the immunoglobulin gene. Germline liverDNA was used as control. (B) Constitutive expression of Stat3 in NPM-ALK tumorcells. Total cell extracts from NPM-ALK� cell lines (lanes 1,2) and from fresh tumorswere immunoblotted with the indicated antibodies. Thymic tumor derived from �cul1transgenic mice served as controls.

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multiple myeloma. In fact, in addition to peripheral plasma celltumors, 20% of our Tg mice displayed primary neoplasms withinthe bone marrow, often involving the dorsal vertebrae. Thesetumors led to the compression and/or infiltration of ganglia andspinal nerves and ultimately resulted in the paralysis of theposterior legs. The clinical presentation and histologic features ofthese tumors closely recapitulated those of human multiple myelo-mas. Therefore, NPM-ALK transgenic mice represent the onlymurine model for multiple myeloma. In fact because the first studyof Anderson et al,38 who demonstrated that after injection ofpristane, Balb/c mice were prone to develop plasmacytomas,several investigators have described several other plasma cell modelsthat do not display the features of human multiple meylomas.32,39-41

The discovery of rare plasmacytoid tumors overexpressingALK in humans16 and the high frequency of plasma cell neoplasmsin our mice strongly indicate that the forced expression of ALKcould transactivate crucial pathways for the development of plasmacell dyscrasias. We and others have shown that ALK can constitu-tively transactivate Stat3. Stat3 is known to play an important rolein the pathogenesis of multiple myelomas, and its activation isrequired for the maintenance and survival of neoplastic plasmacells.42 Interestingly, the activation of Stats might be achieved inmultiple ways. This often occurs via the interleukin 6 receptor(IL-6R) engagement, but recently it was also shown that the

inappropriate activation of fibroblast growth factor receptor 3(FGFR3) can efficiently lead to Stat1 and Stat3 activation.43

Collectively, these findings strongly suggest that the transactivationof Stats, and in particular of Stat3, plays a crucial role in thepathogenesis of plasma cell tumors. Finally, activated ALK viaGrb-2, Shc, and other adaptors leads to the activation of Ras andErk1/2 (data not shown), and Ras has been demonstrated to have animportant role in the pathogenesis of plasma cell neoplasms.44,45

In conclusion our findings have confirmed the tumorigenicactivity of ALK in vivo and have shown that ALK can efficientlytransform T lymphocytes and lead to the development of plasmacell neoplasms. Our model will provide a valuable tool to dissectthe signaling of ALK and to identify new putative recurrentaberrations cooperating with ALK in promoting T-cell transforma-tion. The NPM-ALK mice are the first in vivo murine model formultiple myeloma and represent a unique model in which toinvestigate the efficacy of new therapeutic approach for thetreatment of both ALCL and multiple myelomas.

Acknowledgments

We thank Drs A. Rostagno and E. Zhu for their technical assistance.

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