Translocations Involving Anaplastic Lymphoma Kinase (ALK)

Translocations involving anaplastic lymphoma kinase (ALK)

Justus Duyster*,1, Ren-Yuan Bai1 and Stephan W Morris*,2,3

1Department of Internal Medicine III, Laboratory of Leukemogenesis, Technical University of Munich, Germany; 2Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA; 3Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Anaplastic large-cell lymphoma (ALCL) comprises a al., 2001; Skinnider et al., 1999; Stein et al., 2000). group of non-Hodgkin's lymphomas (NHLs) that were ALCL tumor cells are characterized by the expression ®rst described in 1985 by Stein and co-workers and are of the CD30/Ki-1 antigen. These lymphomas normally characterized by the expression of the CD30/Ki-1 arise from T-cells; in some cases, however, neither T- antigen (Stein et al., 1985). Approximately half of these cell markers nor T-cell receptor rearrangements can be lymphomas are associated with a typical chromosomal detected, leading to a null-cell categorization. Rare translocation, t(2;5)(p23;q35). Much confusion about the cases of ALK-positive ALCL have been reported to be exact classi®cation and clinicopathological features of of B-cell phenotype (Gascoyne et al., 1999); however, this subgroup of NHL was clari®ed with the identi®ca- most investigators do not consider these B-cell tion of NPM ± ALK (nucleophosmin-anaplastic lympho- lymphomas to be classical ALCLs. For example, the ma kinase) as the oncogene created by the t(2;5) (Morris REAL and the new WHO classi®cations exclude B-cell et al., 1994). With the discovery of NPM ± ALK as the cases from the entity of ALCL and place these cases speci®c lymphoma gene mutation, this NHL subtype into a dierent category (diuse large B-cell lympho- could be rede®ned on the molecular level. This ma) (Harris et al., 1994). ALCL arises most frequently achievement was enhanced by the availability of speci®c in children and young adults, but also occurs in older antibodies that recognize ALK fusion proteins in (560 years) patients. These lymphomas have a broad paran-embedded lymphoma tissues. Several excellent morphological spectrum, ranging from cases domi- recent reviews have summarized the histopathological nated by small tumor cells to cases marked by very and molecular ®ndings of ALCL and their use in the large tumor cells, as well as combinations thereof classi®cation of this lymphoma entity (Anagnostopoulos (Benharroch et al., 1998; Falini et al., 1998). A and Stein, 2000; Benharroch et al., 1998; Drexler et al., histological hallmark found in most cases is the 2000; Foss et al., 2000; Gogusev and Nezelof, 1998; common classical ALCL cell, which is characterized Kadin and Morris, 1998; Ladanyi, 1997; Morris et al., by an eccentric localized nucleus with a kidney-like 2001; Shiota and Mori, 1996; Skinnider et al., 1999; shape (Benharroch et al., 1998). The histological Stein et al., 2000). This review will focus on the variability of ALCL led to considerable diculties in molecular function and signal transduction pathways the unequivocal diagnosis of this entity in the past. activated by ALK fusion oncogenes, with recent advances The genes altered in the classical t(2;5) translocation and possible clinical implications to be discussed. were identi®ed and cloned in 1994 (Morris et al., 1994). Oncogene (2001) 20, 5623 ± 5637. Morris et al. (1994) demonstrated that nucleophosmin (NPM) was fused to ALK (anaplastic lymphoma Keywords: ALK; NPM ± ALK; variant ALK fusions; kinase), a previously unknown tyrosine kinase. With ALCL; IMT; pleiotrophin the development of sensitive diagnostic tools to detect ALK, the percentage of CD30+ ALCL cases carrying ALK rearrangements has been rising in recent years ALCL (for a table summarizing the frequency of ALK positivity in ALCL see Morris et al., 2001). Depending ALCL belongs to the group of high-grade NHLs and on the histological subtype (ranging from large cell- to typically presents as an aggressive systemic disease, small cell-predominant), ALK rearrangements can be with or without extranodal involvement (for reviews demonstrated in about 60% of all CD30-positive see Kadin, 1997; Kinney and Kadin, 1999; Morris et ALCL (Morris et al., 2001). Within speci®c ALCL subtypes, 30 ± 50% of pleomorphic ALCL (Kinney et al., 1996; Kinney and Kadin, 1999), more than 80% of *Correspondence: J Duyster, Department of Internal Medicine III, monomorphic ALCL (Kinney et al., 1996), 75 ± 100% Laboratory of Leukemogenesis, Technical University of Munich, of small-cell cases (Falini et al., 1998; Kinney et al., Ismanigerstr 22, 81675 Munich, Germany; E-mail: 1996), and 60 ± 100% of lymphohistiocytic ALCL [email protected]; or SW Morris, Department of (Falini et al., 1998; Pileri et al., 1997) have been shown Pathology, St. Jude Children's Research Hospital, Thomas Tower ± Room 5024, 332 North Lauderdale Street, Memphis, Tennessee TN to be ALK-positive. A higher incidence of ALK- 38105, USA; E-mail: [email protected] positive cases is consistently found in children and Translocations involving ALK J Duyster et al 5624 young adults than older patients (Falini et al., 1999a; localization of the NPM ± ALK protein (Bischof et al., Nakamura et al., 1997; Pulford et al., 1997). In most 1997; Liu and Chan, 1991), the NPM portion of which clinical trials published, ALK-positive lymphomas (so- does not contain any NLS (see section Subcellular called `ALKomas') have had a better prognosis than localizations of ALK fusion proteins). Interestingly, ALK-negative tumors (Nakamura et al., 1997; Sand- several cases of the t(5;17) variant translocation of lund et al., 1994a,b; Shiota et al., 1995). This dierence acute promyelocytic leukemia (APL) have been found in prognosis may be due in part, however, to the to fuse the NPM gene to the retinoic acid receptor younger age of the ALK-positive ALCL population. (RAR) gene (Brunel et al., 1995; Redner et al., 1996). Within this entity, the NPM portion is implicated to be serving in oligomerization with wild-type NPM (Liu Nucleophosmin (NPM)/B23 and Chan, 1991) and in mediating the homo-oligomerization of NPM ± RAR1, therefore contributing to the NPM/B23 is a 38-kD nucleolar protein (Figure 1) aberrant transcriptional activity of this fusion protein encoded on chromosome 5 that contains bipartite (Redner et al., 1996). NPM is involved in yet another nuclear localization signals (NLS) (Michaud and oncogenic fusion, the NPM ± MLF1 chimera formed by Goldfarb, 1991), binds nuclear proteins, and engages the t(3;5) in myelodysplastic syndrome and acute in cytoplasm/nuclear tracking (Borer et al., 1989; nonlymphocytic leukemia (Yoneda-Kato et al., 1996), Szebeni et al., 1995; Valdez et al., 1994). The protein in which the NPM segment mediates NPM ± MLF1 has been suggested to be involved in the assembly and self-association, hetero-association with normal NPM, intranuclear transport of preribosomal particles (Borer and aberrant nucleoplasmic/nucleolar localization of et al., 1989; Yung et al., 1985). Because it is the predominantly cytoplasmic normal MLF1 protein. phosphorylated by casein kinase II in interphase (Chan In the case of NPM ± ALK, the strong ubiquitous et al., 1990) and by p34 cdc2 in mitosis (Peter et al., NPM promoter drives the high-level expression of 1990), an active role for NPM/B23 in the cell cycle has NPM ± ALK in lymphoma cells, and oligomerization been proposed. A decisive involvement of NPM/B23 in mediated by the NPM segment leads to the activation controlling centrosome duplication initiated by CDK2/ of the NPM ± ALK fusion protein. cyclin E-mediated phosphorylation in the events of mitosis was revealed recently. NPM/B23 associates with unduplicated centrosomes and dissociates upon its ALK (anaplastic lymphoma kinase) phosphorylation by CDK2/cyclin E. Thus, NPM/B23 functions as a target of CDK2/cyclin E in the initiation The ALK gene on chromosome 2p23 codes for a of centrosome duplication (Okuda et al., 2000). In cells receptor tyrosine kinase (RTK). Two groups indepen- entering mitosis (while the nucleoli disassemble), NPM/ dently cloned the human and mouse ALK receptor B23 relocates to the cytoplasm at the periphery of the (Iwahara et al., 1997; Morris et al., 1997), which was chromosomes and migrates to the poles of the mitotic shown to code for a protein with a predicted molecular spindle (Ochs et al., 1983; Zatsepina et al., 1999). mass of *180 kD. N-linked glycosylation of ALK NPM/B23 normally undergoes self-oligomerization leads to a glycoprotein that migrates in SDS gels at (Chan and Chan, 1995), as well as hetero-oligomeriza- about 210 kD. ALK has the typical structure of a tion with NPM ± ALK, which leads to the nuclear transmembrane RTK, with a large extracellular

Figure 1 Schematic illustration of the NPM ± ALK fusion protein resulting from the t(2;5) chromosomal translocation. Fusion of the chromosome 5 gene encoding nucleophosmin (NPM) to the chromosome 2 gene encoding anaplastic lymphoma kinase (ALK) results in the expression of a constitutively activated chimeric tyrosine kinase, NPM ± ALK. NPM contains an oligomerization domain (OD; residues 1 ± 117), a metal binding domain (MB; residues 104 ± 115), two acidic amino acid clusters (AD: Asp/Glu-rich acidic domain; residues 120 ± 132 and 161 ± 188) that function as acceptor regions for nucleolar targeting signals, and two nuclear localization signals (NLS; residues 152 ± 157 and 191 ± 197). ALK contains a MAM (Meprin/A5/protein tyrosine phosphatase Mu) domain, a region of about 170 amino acids present in the extracellular portions of a number of functionally diverse proteins that may have an adhesive function (PROSITE database: PDOC 00604, Jiang et al., 1993), in its extracellular segment (ALK residues 480 ± 635). The putative ligand binding site (LBS) for pleiotrophin (ALK residues 391 ± 401) is indicated. TM: transmembrane domain; TK: tyrosine kinase catalytic domain

Oncogene Translocations involving ALK J Duyster et al 5625 domain, a lipophilic transmembrane segment, and a extent in the adult brain. RNA in situ hybridization cytoplasmic tyrosine kinase domain (Figure 1). Only revealed expression in speci®c regions of the developing the cytoplasmic tyrosine kinase domain is fused to brain, with highest expression detectable in thalamus, NPM in NPM ± ALK. mid-brain, olfactory bulb and selected cranial, as well The extracellular domain of ALK is highly similar to as dorsal root, ganglia of mice (Iwahara et al., 1997; the extracellular domain of leukocyte tyrosine kinase Morris et al., 1997). Expression of Alk in hematopoie- (LTK), placing ALK within the family of insulin tic tissues could not be demonstrated. This restricted RTKs. Other RTKs with signi®cant homology to ALK expression pattern of ALK has been con®rmed in include the IGF-1R and cROS (Figure 2). Computer human tissues (Pulford et al., 1997; Shiota et al., analysis has also identi®ed a Drosophila protein, 1994b). The fact that the most abundant expression of suggesting that ALK is an evolutionarily conserved ALK occurs in the neonatal brain suggests a role for tyrosine kinase (Figure 2). Despite their high homol- the receptor in brain development. For example, ALK ogy, the ALK receptor extracellular domain is much may serve as a receptor for a yet unidenti®ed larger than the extracellular segment of LTK (1033 neurotrophic factor. This hypothesis is supported by amino acids (aa) versus 421 aa). Both extracellular the observation that ALK expression partially overlaps domains contain a conserved cysteine-rich EGF-like with the expression of the TRK family of neurotrophin motif and a glycine-rich region (Iwahara et al., 1997; receptors (Barbacid, 1995). Morris et al., 1997). In the part of the extracellular domain present only in ALK, no common motif is A putative ALK ligand, pleiotrophin present except for an amino acid sequence also found in the LDL receptor (ACDFXXDCAXGED) (Iwahara A recent paper describes the identi®cation of pleio- et al., 1997); the function of this motif is unclear. By trophin as a possible ALK ligand (Stoica et al., 2001). Northern blotting, expression of Alk was detected in Pleiotrophin is a polypeptide growth factor that has the murine brain and spinal cord (Iwahara et al., 1997; been shown to induce proliferation in a wide range of Morris et al., 1997). Immunoblotting showed highest cells including epithelial, endothelial and mesenchymal expression in the murine neonatal brain and to a lesser cell lineages (Chauhan et al., 1993; Courty et al., 1991;

Figure 2 Amino acid sequence alignments of NPM ± ALK, hLTK, dALK, hIGF-1R and c-ROS. The tyrosine kinase catalytic domain of NPM ± ALK is shown from aa 178 to 440. hLTK: Human leukocyte tyrosine kinase; dALK: Drosophila homologue of anaplastic lymphoma kinase; hIGF-1R: Human insulin-like growth factor 1 receptor; c-ROS: proto-oncogene c-ROS, a receptor tyrosine kinase related to the viral oncoprotein v-Ros (Neckameyer et al., 1986). ~: ATP-binding site; &: Active site; *: NPM ± ALK tyrosine residues Y338, Y342 and Y343

Oncogene Translocations involving ALK J Duyster et al 5626 Fang et al., 1992; Li et al., 1990; Wellstein et al., 1992). Alk knock-out mice By screening a phage display library with radiolabeled pleiotrophin, a peptide spanning a 10-aa stretch of the Alk knock-out mice have been established in the extracellular domain of ALK could be identi®ed. This laboratory of SW Morris (L Xue and SW Morris, putative pleiotrophin recognition sequence lies within manuscript submitted). These animals have a normal the extracellular part of ALK not shared with LTK lifespan and possess no grossly evident abnormalities. (Figure 1). Thus, pleiotrophin could be a ligand for In addition, no neural defects were observed that ALK but not for LTK. Interestingly, this binding would argue for an essential function of Alk in the region is also conserved in the potential homologue of development of the nervous system. Thus, at present, ALK in Drosophila, suggesting a comparable pleio- the normal function of ALK and its ligand remains an trophin-ALK pathway in the ¯y. In vitro studies open question. demonstrated high anity binding of pleiotrophin to ALK in Farwestern blots (Stoica et al., 2001). By Scatchard analysis, a Kd value of approximately 35 pM NPM ± ALK was calculated. A cell line overexpressing ALK showed enhanced growth in soft agar upon pleiotrophin The t(2;5) chromosomal rearrangement leads to the stimulation. In addition, pleiotrophin induced auto- expression of an 80-kD fusion protein containing the phosphorylation of ALK following stimulation, and ®rst 117 aa of NPM fused to the C-terminal residues phosphorylation of several potential ALK substrates 1058 ± 1620 of ALK (Fujimoto et al., 1996; Morris et including IRS-1, SHC, PLC-gamma and p85 could be al., 1994). The reciprocal ALK ± NPM fusion gene is demonstrated. While these data convincingly argue for not transcribed at signi®cant levels and thus does not a functional link between pleiotrophin and ALK, contribute to the molecular pathogenesis of ALCL several questions remain including whether ALK is (Beylot-Barry et al., 1998; Cordell et al., 1999; Delsol indeed the sole receptor for pleiotrophin. The over- et al., 1997; Morris et al., 1994). The chromosomal lapping expression patterns of ALK and pleiotrophin break within the ALK genomic sequence occurs in a in the nervous system, including the thalamus and 1935-bp intron that is located between the exons midbrain in the adult and developing brain, suggest encoding the transmembrane and juxtamembrane that the molecules indeed could represent a growth domains of ALK. The chromosomal break within the factor/receptor pair (Schulte and Wellstein, 1997). NPM genomic sequence occurs in intron 4 of NPM However, pleiotrophin eects have been demonstrated (Ladanyi and Cavalchire, 1996a; Chan et al., 1997; also in endothelial cells and ®broblasts (Chauhan et al., Luthra et al., 1998). Interestingly, all fusion proteins 1993; Courty et al., 1991; Fang et al., 1992; Li et al., containing rearranged ALK identi®ed so far contain 1990; Wellstein et al., 1992). Elevated pleiotrophin exactly the same 563 aa comprising the cytoplasmic tail levels in the serum of patients suering from a variety of ALK. Only a single ALCL carrying a variant of solid tumors have been demonstrated (Fang et al., breakpoint has been described to date (with the break 1992; Souttou et al., 1998) and animal studies have located in the ALK exon coding for the juxtamembrane suggested that pleiotrophin may contribute to tumor region), leading however to an essentially identical growth, invasion and metastasis (Chauhan et al., 1993; fusion protein product (Ladanyi and Cavalchire, Choudhuri et al., 1997; Czubayko et al., 1994, 1996; 1996b). These observations suggest that the ALK Schulte et al., 1996). However, ALK expression has not sequences present in NPM ± ALK are the minimum been detected in any of these dierent cell types in mice residues of the protein required to lead to ALCL. or humans. The paper by Stoica et al. (2001) suggests NPM ± ALK is a potent oncogene capable of for the ®rst time that ALK expression may occur in transforming a wide array of dierent cell types in some solid cancer cell lines and NIH3T3 cells. The vitro, including mouse and rat ®broblasts like expression of ALK was correlated with the responsive- NIH3T3, Fr3T3 and Rat-1 cells, and hematopoietic ness to pleiotrophin in these cells. However, ALK cell lines such as the myeloid line 32Dcl3 and the expression could not be demonstrated by immunoblot- lymphocytic cell line Ba/F3 (Bai et al., 1998b; ting with anti-ALK antibodies, the detection requiring Bischof et al., 1997; Fujimoto et al., 1996; Kuefer et an RT ± PCR procedure with 30 cycles of ampli®cation al., 1997; Mason et al., 1998). In addition, primary followed by hybridization with a radiolabeled internal mouse bone marrow cells can be eciently trans- oligonucleotide. Thus, ALK receptor density seems to formed by NPM ± ALK in vitro (Bai et al., 2000). In be extremely low. It seems surprising that the profound comparison to other oncogenic fusion proteins such as and multiple eects of pleiotrophin might be mediated BCR ± ABL, NPM ± ALK possesses strong transform- solely by the ALK receptor. Indeed, other cellular ing potential in both hematopoietic and ®broblast cell receptors for pleiotrophin have been identi®ed, protein lines. For example, soft-agar colonies obtained by tyrosine phosphatase zeta/RPTP beta and syndecan-3, transfection with NPM ± ALK grow faster and are that may mediate some of these eects (Maeda et al., larger than colonies transformed by BCR ± ABL 1996; Raulo et al., 1994). In addition, further studies (unpublished observation). Given the demonstration are required to establish pleiotrophin as the sole ligand that NPM ± ALK-transduced bone marrow trans- for the ALK receptor, and the physiological relevance planted into irradiated recipient mice can induce a of this receptor/ligand pair. lymphoma-like disease, it is reasonable to assume that

Oncogene Translocations involving ALK J Duyster et al 5627 NPM ± ALK is the causative oncogene in ALK- positions 5, 7 and 9 of normal NPM are essential for positive ALCL (Kuefer et al., 1997). oligomer formation (Liu and Chan, 1991). An NPM ± The t(2;5) brings the truncated ALK gene under the ALK mutant in which these residues were mutated to control of the strong NPM promoter, leading to the leucines was still capable of forming homo-oligomers, high-level expression of the NPM ± ALK fusion protein as shown in both sucrose gradient and SDS ± PAGE in cells. NPM ± ALK-positive cells show abundant analysis (Bischof et al., 1997). This mutant also tyrosine phosphorylation of NPM ± ALK itself as well retained the ability to transform ®broblasts. Thus, the as a number of other proteins, even when the cells are NPM sequence requirements for homo-oligomerization cultured in the absence of growth factors (Bai et al., may dier somewhat between NPM ± ALK and NPM. 1998b; Bischof et al., 1997; Fujimoto et al., 1996; The exact motif and the mechanism of homo- Shiota et al., 1994a). The fusion of NPM to the oligomerization of NPM ± ALK are still incompletely cytoplasmic tyrosine kinase domain of ALK leads to understood and require further investigation. constitutive activation of the ALK tyrosine kinase NPM sequences could serve solely as an oligomer- function. It is known that the normal NPM protein ization motif, with no further essential functions for forms oligomers, although a classical coiled-coil the transforming activity of NPM ± ALK, or they could domain could not be identi®ed. The fusion of the N- potentially serve additional required roles. To address terminal oligomerization motif in NPM, or function- this issue, ALK chimeras have been constructed in ally similar motifs in the alternative ALK fusion which the NPM part of NPM ± ALK was replaced with partners, to the cytoplasmic tyrosine kinase domain unrelated domains known to mediate oligomerization of ALK leads to activation of the kinase catalytic (Bischof et al., 1997). The TPR (translocated promoter domain, mimicking oligomerization of the receptor by region) protein is a nuclear pore protein that contains a natural ligand. While activation of a given receptor two leucine zipper motifs, and is rearranged in the by a ligand occurs only transiently (being terminated TPR ± MET oncogene (Rodrigues and Park, 1993). A rapidly by cellular internalization and receptor degra- TPR ± ALK chimera displayed full transforming func- dation), oligomerization of oncogenic fusion kinases tion in ®broblasts. The oncogenic activation of ALK leads to unregulated, constitutive activation. The by a fusion partner with no homology to NPM, such commonly accepted paradigm is that oncogenic as TPR, supports a singular function for the NPM tyrosine kinases recruit, and in turn constitutively residues in NPM ± ALK of mediating oligomerization. activate, promitogenic signaling cascades, leading to the transformation of cells. By recruiting SRC homology 2 (SH2) or phosphotyrosine binding (PTB) Subcellular localizations of ALK fusion proteins domain-containing molecules, speci®c signaling pathways are activated. NPM ± ALK is a highly autophos- The localization of a portion of the total cellular phorylated molecule and contains 21 putative auto- NPM ± ALK protein within the nuclear compartment is phosphorylation sites that could serve as docking unexpected, given that the NPM nuclear localization regions for SH2- or PTB-containing molecules. motifs are not retained in the fusion. As mentioned above, NPM is a protein involved in transport processes between the nucleolus and the cytoplasm Role of NPM and is able to oligomerize, being present in cells The normal NPM protein is able to form hexamers, primarily as hexamers. Thus, it is likely that the partial and this oligomerization ability leads to the consti- nuclear localization of NPM ± ALK is due to its tutive activation of NPM ± ALK However, no classical association with endogenous nuclear-localized NPM. oligomerization motifs are present in NPM ± ALK. To Indeed, such an association has been demonstrated in further study the domains in NPM ± ALK required for the NPM ± ALK-positive cell line SUP-M2 by both self-association, several NPM deletion mutants have coimmunoprecipitation and sedimentation gradient been constructed (Bischof et al., 1997). Deletion of the experiments (Bischof et al., 1997). D64NPM ± ALK, ®rst 64 aa of NPM (D64NPM ± ALK), which have been D65 ± 103NPM ± ALK and DMBNPM ± ALK are all unable reported to mediate hexamer formation of the normal to oligomerize with endogenous NPM and show the protein (Liu and Chan, 1991), still retains autokinase anticipated cytoplasmic localization. ability in vitro but lacks kinase activity in vivo. The nuclear localization of NPM ± ALK does not Consequently, this mutant does not possess transform- seem to be required for lymphomagenesis. The chimeric ing abilities in cell lines, as judged by focus formation TPR ± ALK protein, although displaying full transform- or soft-agar growth (Bischof et al., 1997). Loss of ing capabilities in ®broblasts, shows an exclusive transforming ability was also observed with deletion of cytoplasmic location (Bischof et al., 1997). This assump- NPM residues 65 ± 103 (D65 ± 103NPM ± ALK) and upon tion is also supported by the fact that the majority of the deletion of NPM residues 104 ± 114, reported to recently identi®ed alternative ALK fusion proteins are comprise a putative metal binding motif (DMBNPM ± localized solely to the cytosol (see Figure 5). ALCLs ALK) (Chan et al., 1989). These data suggested that bearing alternative ALK gene rearrangements are the complete 117-aa portion of NPM must be present indistinguishable from ALCLs with the classical t(2;5). in NPM ± ALK for its transforming function. Earlier The fact that a variety of oligomerization domain- studies had indicated that methionine residues at containing proteins can replace NPM and the exclusively

Oncogene Translocations involving ALK J Duyster et al 5628 cytoplasmic location of these ALK fusion variants mutagenesis failed to iden®ty a single tyrosine residue together support the notion that only the cytoplasmic in NPM ± ALK responsible for the recruitment of pool of NPM ± ALK contributes to oncogenesis. GRB2 (Bai et al., 1998b; Fujimoto et al., 1996). Complex formation between GRB2 and NPM ± ALK therefore may involve multiple tyrosine residues or Signaling pathways activated by NPM ± ALK perhaps intermediate proteins. It is possible that NPM ± ALK(Y567F), which is unable to recruit SHC, Structural homology studies place NPM ± ALK into is still capable of activating the promitogenic RAS the family of insulin RTKs, with the highest homology pathway by recruiting GRB2. Thus, the importance of to LTK. The two proteins share 64% aa identity in the RAS pathway for NPM ± ALK-mediated oncogeni- their kinase catalytic domains (Figure 2) (Fujimoto et city has to be further evaluated. al., 1996; Iwahara et al., 1997; Morris et al., 1997). LTK has been demonstrated to activate the RAS ± PLC-gamma MAP kinase pathway by recruiting the adaptor molecule SHC (Ueno et al., 1996). In addition, IRS-1 Additional studies have investigated the importance of has been shown to be recruited by LTK and seems to autophosphorylation of NPM ± ALK by site-directed be of importance in delivering an antiapoptotic signal mutagenesis of each of the possible autophosphorylation by this tyrosine kinase (Ueno et al., 1997). These sites in the protein (Figure 3) (Bai et al., 1998b). ®ndings prompted investigators to elucidate the role of Interestingly, individual mutation of each of three these and other adaptor and signaling proteins for the tyrosine residues, Tyr338, Tyr342 and Tyr343, led to a mitogenicity induced by NPM ± ALK. severe impairment of NPM ± ALK tyrosine kinase activity. These tyrosine residues are located within the kinase catalytic domain of NPM ± ALK close to the RAS pathway catalytic core (Figure 2) and it is likely that mutation of Analysis of the ALK sequence revealed two putative these sites interferes with the ability of NPM ± ALK to binding sites for PTB domains, NPNY156 and catalyze phosphorylation. All other individual tyrosine- NPTY567; subsequently, it was shown that the adaptor to-phenylalanine mutations retained kinase activity molecules IRS-1 and SHC bind to these sites, (Figure 3). To evaluate the importance of dierent respectively, probably via their PTB domains (Bischof tyrosine residues for the mitogenicity of NPM ± ALK, all et al., 1997; Fujimoto et al., 1996). SHC is an mutants were tested for their ability to induce IL-3- important signal transducer known to couple growth independent growth in Ba/F3 cells, a pro-B cell line that factor receptors such as the EGF or insulin receptors requires IL-3 for survival and growth. Induction of IL-3 to the mitogenic RAS pathway. SHC binds directly to independence in this cell line is an indication of the the activated EGF receptor and then forms a complex transforming/mitogenic potency of a particular NPM ± with GRB2 and SOS (mammalian homologue of ALK mutant. Interestingly, only one mutant that Drosophila son of sevenless), leading to the activation retained kinase activity failed to induce stable IL-3- of RAS. IRS-1 is involved in signal transduction from independent growth, NPM ± ALK(Y664F). Subse- the insulin receptor. Site-directed mutagenesis has quently, Tyr664 was shown to recruit PLC-gamma, con®rmed that Tyr156 and Tyr567 in NPM ± ALK which leads to enhanced IP3 production in NPM ± ALK- are indeed responsible for the complex formation of expressing cells (Bai et al., 1998b). Mutation of Tyr664 NPM ± ALK with IRS-1 and SHC, respectively. abolishes binding and activation of PLC-gamma by However, NPM ± ALK mutants defective in recruiting NPM ± ALK. Overexpression of wild-type PLC-gamma these molecules still show transforming potential in could partially restore the transforming function of the NIH3T3 cells and in the pro-B cell line Ba/F3, NPM ± ALK(Y664F) mutant. The PLC-gamma path- suggesting that these signaling cascades are not way seems to be of general importance for NPM ± ALK- essential for the transforming function of the chimeric mediated mitogenicity since NPM ± ALK(Y664F) also protein in these cells (Figure 3) (Bai et al., 1998b; showed impaired transforming capabilities in NIH3T3 Fujimoto et al., 1996). The importance of these cells. The fact that all mutants retaining kinase activity signaling pathways for the oncogenicity of NPM ± except NPM ± ALK(Y664F) still rendered Ba/F3 cells ALK, however, has not been evaluated in more growth factor-independent emphasizes the importance de®nitive in vivo models of ALCL (see section Animal of PLC-gamma signaling for the transforming function models of ALCL). Thus, whether these signaling of the fusion protein (Figure 3). However, as is the case pathways are required for the transforming ability of for the other signaling molecules mentioned here, NPM ± ALK in vivo remains unanswered. veri®cation of the signi®cance of the PLC-gamma Besides SHC and IRS-1, GRB2 has also been pathway in an in vivo model of ALCL still remains to demonstrated to coprecipitate with NPM ± ALK in be demonstrated. ALCL cells (Fujimoto et al., 1996). GRB2 binds NPM ± ALK in a GST pull-down assay even under PI-3 kinase denaturing conditions, suggesting a direct interaction between the GRB2 SH2 domain and a phosphoty- NPM ± ALK(Y664F)-expressing Ba/F3 cells do not rosine residue on NPM ± ALK. However, site-directed stably grow without IL-3. However, in contrast to

Oncogene Translocations involving ALK J Duyster et al 5629

Figure 3 Tyrosine-to-phenylalanine mutations of the putative autophosphorylation sites of NPM ± ALK and their biological impact. Wild-type NPM ± ALK or mutants were transfected and stably selected in IL3-dependent Ba/F3 cells. Dependency on IL-3 was tested by culturing the cells at 105/ml in IL3-free RPMI 1640 medium supplemented with 5% FCS for 3 weeks (Bai et al., 1998b). The identi®ed binding sites of IRS-1, SHC and PLC-gamma are indicated (Bai et al., 1998b; Fujimoto et al., 1996) parental Ba/F3 cells or Ba/F3 cells expressing a kinase- It is well documented that apoptosis protection in Ba/ defective mutant of NPM ± ALK, NPM ± ALK F3 cells by IL-3 is mediated in part by activation of PI-3 (Y664F) cells do not undergo complete apoptosis after kinase (phosphatidylinositol-3 kinase)/AKT and subse- IL-3 withdrawal and re-addition of IL-3 within a week quent phosphorylation and inactivation of the proapop- can rescue these cells (Bai et al., 1998b). This observation totic molecule BAD. A widely distributed isoform of PI- suggested that PLC-gamma is predominantly required 3 kinase is a heterodimer of a 110-kd catalytic subunit for the promitogenic properties of NPM ± ALK and for and an 85-kd (p85) regulatory subunit, which is found in its ability to replace IL-3 for mitogenicity but not for the cellular complexes with ligand-activated growth factor protection against apoptosis. receptors and non-receptor tyrosine kinases (Cantley et

Oncogene Translocations involving ALK J Duyster et al 5630 al., 1991; Leevers et al., 1999). Speci®c phosphorylated LTK, in contrast to NPM ± ALK, has been shown to tyrosine residues present on the tyrosine kinase and the bind directly to the p85 subunit of PI-3 kinase. two SH2 domains of p85 mediate formation of these Mutation of the p85-binding phosphotyrosine residue complexes. The association of p85 with activated within LTK results in loss of the antiapoptotic function tyrosine kinases is believed to be sucient for the of an EGF ± LTK chimear in Ba/F3 cells (Ueno et al., activation of the catalytic function of the p110 subunit. 1997). Thus, PI-3 kinase seems to be another important This activation leads to phosphorylation of the D-3 player in the signaling events that lead to NPM ± ALK- position of phosphatidylinositol (PI), phosphatidylino- mediated transformation (summarized in Figure 4). sitol 4-phosphate (PI4P) and PI4,5P (Leevers et al., 1999). The 3-phosphoinositide that is generated is able to STAT (signal transducer and activator of bind to pleckstrin homology (PH) domains of signaling transcription) proteins molecules and to activate various downstream molecules, including the serine/threonine kinase AKT/PKB. Additional signaling molecules that have recently been AKT in turn is capable of preventing caspase-9 reported to be of relevance for the genesis of NPM ± activation by maintaining mitochondrial integrity, ALK-positive lymphomas include STAT3 and STAT5. phosphorylating and inactivating the proapoptotic For example, NPM ± ALK has been shown to activate BCL-2 family member BAD, activation of NF-kB, and STAT5 and this activation may be essential for reduction of the transcription of the Fas ligand, all of lymphomagenesis (Nieborowska-Skorska et al., 2000). which contribute to its antiapoptotic function (Khwaja, Retroviral infection of NPM ± ALK-positive cells with 1999). a dominant-negative STAT5B mutant inhibited the Two groups have reported the activation of PI-3 anti-apoptotic activity of NPM ± ALK in these studies. kinase by NPM ± ALK (Bai et al., 2000; Slupianek et al., 2001). Blocking the activation of PI-3 kinase with Role of CD30 speci®c inhibitors like wortmannin or LY294002 was demonstrated to prevent NPM ± ALK-mediated mito- Most, if not all, NPM ± ALK-positive lymphomas genicity to a much greater extent than IL-3-mediated express the CD30 transmembrane receptor protein. mitogenicity in Ba/F3 cells. PI-3 kinase inhibitors were Therefore, it is intriguing to speculate that CD30 and also able to induce apoptosis in the ALCL cell line JB6 NPM ± ALK are functionally connected, and that both (Bai et al., 2000). Expression of dominant-negative p85 molecules may be required for the development of or AKT mutants induced apoptosis in NPM ± ALK- ALCL. In support of this notion, it was ®rst shown in expressing cell lines and prevented tumor growth in 1994 that CD30 can physically interact with NPM ± syngeneic mice (Slupianek et al., 2001). Furthermore, ALK (Shiota et al., 1994a). BAD-induced apoptosis in COS cells could be partially CD30 belongs to the TNF receptor superfamily and blocked by the overexpression of NPM ± ALK (Bai et it is expressed on activated T-cells and on tumor cells al., 1998a). It is important to note that AKT activation of hematopoietic origin, including the neoplastic cells was also demonstrated in ALCL patient samples of Hodgkin's disease and ALCL (Ellis et al., 1993; (Slupianek et al., 2001). The complex of NPM ± ALK Schwab et al., 1982; Stein et al., 1985). Stimulation of and p85 is mediated mainly by the C-terminal SH2 CD30 by its ligand, CD30L, has been shown to elicit a domain of p85, with the N-terminal SH2 domain not plethora of cellular eects, including both proliferative apparently important for the interaction (Bai et al., and apoptotic responses in dierent cell types (Lee et 1998a). In an eort to identify the binding site of the al., 1996; Smith et al., 1993). It has been proposed that p85-CSH2 domain on NPM ± ALK, Tyr418 was shown the TNF receptor superfamily can either induce to be a potential interaction site in vitro; however, apoptosis by recruitment of molecules like FADD mutation of this site failed to abolish binding in and TRADD, leading to a caspase activation and coimmunoprecipitation experiments using whole cell apoptosis, or promote survival through the recruitment extracts. The most likely explanation for these ®ndings of TRAF proteins and activation of NF-kB (Beg and is that an indirect association occurs between NPM ± Baltimore, 1996; Hsu et al., 1996; Liu et al., 1996; Van ALK and p85 through an additional adaptor mole- Antwerp et al., 1996). However, in contrast to other cule(s). For example, IRS-1 could recruit p85 to TNF receptor family members, CD30 lacks the so- NPM ± ALK. However, mutation of the IRS-1 binding called death domain that is required for the recruit- site (NPM ± ALK Y156F) failed to reduce the binding ment of molecules such as FADD and TRADD that anity of NPM ± ALK for p85. Given the numerous are responsible for caspase activation (Durkop et al., adaptor molecules that can function as linkers between 1992). Nevertheless, like other TNF receptor family tyrosine kinases and SH2 domain-containing eector members, CD30 induces pleiotropic eects in cells molecules, a complex involving p85, NPM ± ALK, and upon its activation. Recently, it was observed that an adaptor molecule is likely (Pawson and Gish, 1992). CD30L induces cell death in NPM ± ALK-positive Indeed, a simultaneous complex among NPM ± ALK, Karpas299 cells but not in an NPM ± ALK-negative p85 and Gab2, SHC or CrkL has been demonstrated Hodgkin's disease-derived cell line, HDLM-2 (Hubin- (Bai et al., 2000). Thus, these adaptor molecules are ger et al., 1999a). These studies also demonstrated that candidates linking NPM ± ALK via the regulatory CD30 forms a complex with NPM ± ALK in Kar- subunit p85 to the antiapoptotic PI-3 kinase pathway. pas299 cells (Hubinger et al., 1999a). However, no

Oncogene Translocations involving ALK J Duyster et al 5631

Figure 4 Schematic illustration of signaling cascades activated by NPM ± ALK de®nite functional relationship between the two an open issue and requires further investigation. The proteins could be demonstrated, although it was fact, however, that stimulation of CD30 leads to speculated that CD30 may target NPM ± ALK to the apoptotic death of ALCL cells provides a rationale inner face of the cell membrane, allowing the for the use of CD30-activating antibodies in therapeu- phosphorylation of proteins in this region and perhaps tic approaches. Indeed, in animal models, this contributing to the apoptotic response observed after approach has shown potentially promising results CD30 activation in NPM ± ALK-positive cells. Another (Pfeifer et al., 1999; Tian et al., 1995). paper recently con®rmed the proapoptotic eect of CD30L on ALCL cells (Mir et al., 2000). It was demonstrated in this study that CD30 activation leads Other oncogenic fusions involving ALK to the induction of apoptotic death of ALCL cells, with the selective reduction of TNF receptor-associated Immunohistochemical staining has revealed that about factor 2 (TRAF2) function and impairment in the 15 ± 25% of ALK-positive ALCLs do not exhibit the ability of the cells to activate the pro-survival typical staining pattern of NPM ± ALK in both the transcription factor NF-kB(Miret al., 2000). Again, nucleus and cytoplasm, but rather possess exclusively however, no clearcut connection of these eects to the cytoplasmic staining (Benharroch et al., 1998). Variant expression of NPM ± ALK was demonstrated. chromosomal translocations involving the ALK locus at Thus, the molecular and functional relationship of chromosome 2p23 have also been observed, suggesting the coexpression of CD30 and NPM ± ALK in ALCL the existence of ALK fusion partners other than NPM. cells remains enigmatic. At present, it is unclear In addition, immunoblotting with ALK- and NPM- whether the expression of CD30 contributes to the speci®c antibodies has revealed variant ALK proteins of development of ALCL at the molecular level. Rather, 85-, 97-, 104- and 113-kD in ALCL cases containing it may be that the chromosomal translocations leading t(2;3)(p23;q21) and t(1;2)(q21;p23), and these proteins to the expression of ALK fusion proteins occur at the did not contain NPM (Pulford et al., 1999). stage of a speci®cally dierentiated T-cell that happens Over the past 3 years, a number of variant ALK to express CD30. Thus, CD30 expression would be an rearrangements have been cloned, and the ALK fusion epiphenomenon, and not functionally related to the partners identi®ed (Figure 5). Some of these rearrange- development of ALCL. The fact that NPM ± ALK is ments have been observed only in ALCL cases, such as able to transform CD30-negative hematopoietic cell NPM ± ALK itself, ATIC ± ALK, TFG ± ALKL and lines and that lymphomas induced by NPM ± ALK in TFG ± ALKS, while others have been found in both mice are CD30 negative also argue against a functional ALCL and in¯ammatory myo®broblastic tumors role for CD30 in the development of ALK-positive (IMTs), like TPM3 ± ALK and CLTC ± ALK. Finally, ALCL. Nonetheless, whether CD30 functionally con- two rearrangements have been seen to date in IMT only, tributes to the molecular pathogenesis of ALCL is still RanBP2 ± ALK and TPM4 ± ALK. In all of these variant

Oncogene Translocations involving ALK J Duyster et al 5632 fusions, NPM is replaced by a protein that contains some has been demonstrated. As for TPM3 ± ALK, fusion form of oligomerization motif, suggesting that the with TPM3 leads to the constitutive activation of the mechanism underlying ALK tyrosine activation is truncated NTRK1 (Butti et al., 1995). Like most similar in all of these chimeric proteins and that the variant ALK fusions, TPM3 ± ALK is located exclu- signal transduction pathways activated are identical. sively in the cytoplasm of lymphoma cells. This hypothesis is supported by the fact that the clinico- The t(2;3)(p23;q21) translocation leads to the pathological features of ALCL containing alternative formation of two dierent fusion proteins, TFG ± ALK chromosomal rearrangements seem to be indis- ALKL and TFG ± ALKS, depending upon the chro- tinguishable from those with the classical t(2;5) (Falini et mosomal breakpoint in the TFG gene (Hernandez et al., 1999b). The prognosis of these variant ALK-positive al., 1999). TFG stands for TRK-fused gene and was lymphomas seems comparable to NPM ± ALK-positive originally cloned as a fusion partner for NTRK1 in ALCL and is signi®cantly better than ALK-negative papillary thyroid carcinomas (Greco et al., 1995). As cases. In addition, like NPM ± ALK-positive tumors, all for TPM3, TFG contains a coiled-coil domain that can variant ALK-positive cases display a T- or null-cell mediate the oligomerization and constitutive activation phenotype, show comparable nodal and extranodal of the fused tyrosine kinase. involvement, and occur predominantly in children and Three groups independently identi®ed the ALK young adults (Morris et al., 2001). fusion partner involved in the inv(2)(p23q35), ATIC, TPM3 ± ALK is expressed from a t(1;2)(q25;p23) which encodes 5-aminoimidazole-4-carboxamide ribo- translocation (Lamant et al., 1999). This translocation nucleotide formyltransferase/IMP cyclohydrolase, the leads to the fusion of the N-terminal 221 residues of enzyme responsible for the ®nal steps of de novo purine TPM3 to the cytoplasmic portion of ALK. TPM3 is a nucleotide biosynthesis (Colleoni et al., 2000; Ma et al., nonmuscle tropomyosin that contains an N-terminal 2000; Trinei et al., 2000). The N-terminal 229 residues coiled-coil structure, allowing its self-association and of ATIC are fused to ALK and are capable of leading leading to the activation of the TPM3 ± ALK fusion to homo-oligomerization and constitutive activation of protein. TPM3 has been shown to be involved in other the ATIC ± ALK chimeric kinase. chromosomal translocations as well; for example, in The largest variant ALK fusion protein identi®ed thus colon and papillary thyroid cancers, a fusion of TPM3 far, CLTC ± ALK, is expressed from the t(2;17)(p23;q23) with the truncated NTRK1 receptor tyrosine kinase (Touriol et al., 2000). CLTC is the heavy chain of the

Figure 5 ALK fusion proteins, the chromosomal rearrangements that generate them, and their frequency and subcellular localization. C: cytosolic; N: nuclear; NM: nuclear membrane. The numbers above the fusion proteins indicate the length (aa) of the fusion partners incorporated into each chimeric protein, and the total number of residues in each ALK fusion

Oncogene Translocations involving ALK J Duyster et al 5633 clathrin molecule, which is composed of both light and ALK-positive lymphomas, these B-cell tumors occur heavy chains and is found in the coated vesicles predominantly in adults and have a poor outcome. responsible for the intracellular transport of molecules. Whether the full-length ALK receptor is constitutively Clathrin proteins normally form trimolecular complexes, activated or functionally contributes to this type of indicating that they are able to oligomerize. In contrast lymphoma is at present unclear. to other variant ALK fusions, CLTC ± ALK does not show a diuse cytoplasmic localization but rather ALK expression in inflammatory myofibroblastic exhibits a ®ne granular staining pattern that is presumed tumors (IMTs) to be due to the binding of CLTC ± ALK to clathrin- containing vesicles. CLTC ± ALK was initially misiden- In¯ammatory myo®broblastic tumors (IMTs) possess a ti®ed as involving the closely related CLTCL clathrin pseudosarcomatous in¯ammatory appearance and can heavy chain gene located at chromosome 22q11.2 occur in nearly every soft tissue and viscera of the body (Touriol et al., 2000). (Morris et al., 2001). These tumors mainly aect Two additional ALK rearrangements have been children and young adults and histopathologically are cloned in cases of ALK-positive in¯ammatory myo- composed of myo®broblasts together with in®ltrating ®broblastic tumors (IMTs). The t(2;19)(p23;p13.1) lymphocytes, eosinophils and plasma cells. The diag- initiates the expression of a TPM4 ± ALK fusion protein, nosis is often dicult depending on the relative TPM4 being a nonmuscle tropomyosin highly related to contribution of the myo®broblastic versus in¯amma- the TPM3 protein described above (Law-rence et al., tory components. Most IMTs can be cured by surgical 2000). Another novel ALK fusion results from a resection of the tumor; however, cases of malignant, t(2;2)(p23;q11-13) or inv(2)(p23q11-13) (SW Morris, invasive and metastatic IMTs have been described. manuscript submitted). Ran binding protein 2 (RanBP2) Recently, chromosomal rearrangements involving band (Mahajan et al., 1997), also known as Nup358, is a large 2p23 were identi®ed in IMT and it was demonstrated nucleopore protein encoded at chromosome 2q11-13 that these rearrangements lead to the generation of that is localized at the cytoplasmic side of the nuclear ALK fusion proteins such as TPM3 ± ALK and pore complex. The N-terminal 867 residues of this TPM4 ± ALK (Grin et al., 1999; Lawrence et al., protein are fused to ALK in RanBP2 ± ALK. In contrast 2000). IMT cases that express CLTC ± ALK and to the other variant ALK fusion proteins, RanBP2 ± RanBP2 ± ALK have also been demonstrated, and ALK displays a nuclear membrane staining pattern that immunostaining studies of 68 IMTs showed ALK is thought to be due to association of the fusion with expression in 42 cases (61.8%) (SW Morris, manuscript normal RanBP2 at the nuclear pores. submitted). This high frequency of involvement Interestingly, all of the variant rearrangements cloned suggests that ALK fusions are of pathogenic impor- thus far contain the same breakpoint within the ALK tance on the molecular level for this neoplasm. gene, suggesting that this chromosomal region is very susceptible to breakage. Alternatively, shorter fragments Neuroblastoma of ALK may not be sucient to induce lymphoma in vivo. This would suggest that the N-terminal sequences ALK expression detectable by immunoreactive of the ALK cytoplasmic segment outside of the catalytic methods is normally restricted to neural tissues. A domain must be present and are of importance for recent paper showed that in cell lines derived from lymphomagenesis. Yet additional variant ALK fusions neuroblastomas and neuroectodermal tumors approxi- may be identi®ed in the future. For example, a number of mately 45% express full-length ALK (Lamant et al., rearrangements in ALCL involving 2p23 with loci other 2000). In primary neuroblastomas, ALK expression than 5q35 remain in which fusion partners have not yet was demonstrated in 92% of the cases examined. been cloned, including t(1;2)(q21;p23), t(2;2)(p23;q23), However, no correlation between ALK expression and t(2;13)(p23;q34) and t(2;22)(p23;q11.2) (Ladanyi, 1997; known prognostic factors for neuroblastoma could be Mitev et al., 1998; Park et al., 1997; Rosenwald et al., identi®ed. There was also no evidence of constitutive 1999). activation of the full-length ALK receptor in these tumors (although in vivo exposure to ligand would be expected to produce regulated, but contextually aberrant, receptor activation). Therefore, con®rmation Other diseases associated with ALK expression of a pathophysiological role for ALK in the development of neuroblastoma currently requires additional Large B-cell lymphomas with full-length ALK expression study. Rare cases of large B-cell lymphoma have been described that express the full-length ALK receptor NPM ± ALK expression in normal cells (Delsol et al., 1997). PCR and FISH failed to identify ALK rearrangements in these cases and the mechanism Using a highly sensitive RT ± PCR procedure, NPM ± of full-length ALK expression in these lymphomas is ALK expression has been demonstrated in peripheral unknown. The tumor cells in these lymphomas lack blood cells of healthy individuals (Trumper et al., CD30 expression but do express the epithelial 1998). As has been previously shown for the t(9;22) membrane antigen (EMA). In contrast to NPM ± and BCR ± ABL, this observation suggests that t(2;5)

Oncogene Translocations involving ALK J Duyster et al 5634 chromosomal rearrangements do occur in cells other lymphoma cells from translating the NPM ± ALK than the lymphoma-inducing target cell of NPM ± fusion protein. Such an approach has shown promising ALK. It is likely that the detection of NPM ± ALK in results in vitro (HuÈ binger et al., 1999b). However, this cases of CD30-positive Hodgkin's disease, which has technique is hampered by the diculties of introducing been very controversially discussed in the past, may be ribozymes into cells, and cell-permeable ribozymes due to the detection of NPM ± ALK in otherwise against NPM ± ALK have not yet been employed normal bystander cells. successfully. In addition, NPM ± ALK-speci®c ribozymes only showed antiproliferative eects when highly overexpressed in NPM ± ALK-positive cells (HuÈ binger Animal models for ALCL et al., 1999b). Thus, while certainly promising, this approach has signi®cant limitations that must be To study the in vivo oncogenicity of NPM ± ALK, overcome to be feasible. transplantations of retrovirally infected bone marrow Another therapeutic approach involves the inhibition into mice have been performed. For example, Kuefer et of signal transduction pathways known to be crucial al. (1997) demonstrated the development of B-lineage for NPM ± ALK-mediated oncogenicity. Along this lymphomas with a 4 ± 6 month latency in BALB/cByJ line, inhibition of PLC-gamma has been shown to be mice, using the pSRaMSVtkneo construct to direct quite eective in preventing the proliferation of cell NPM ± ALK expression. Tumors arose in the mesen- lines engineered to express NPM ± ALK, as well as cell teric lymph nodes, with metastases to the lungs, lines derived from NPM ± ALK-positive ALCL pa- kidneys, liver, spleen, and paraspinal area. More tients (Bai et al., 1998a). However, since NPM ± ALK recently, lymphomas have been successfully induced is capable of activating several promitogenic and in BALB/c mice after a latency of approximately 6 antiapoptotic pathways, inhibition of only one of these weeks using an MSCV-based EGFP bicistronic vector pathways may not be sucient to completely eradicate that programs high-level expression in hematopoietic the malignant clone in ALCL. Thus, inhibition of stem cells (Miething et al., 2000). A massive in®ltration NPM ± ALK at the very beginning of the signaling of NPM ± ALK-positive cells in the spleen, lymph cascade ± i.e., of the constitutively activated tyrosine nodes, and bone marrow could be observed in these kinase itself ± may be more ecient. Indeed, tyrosine animals. More interestingly, cytologic examination kinase inhibitors have gained much attention upon the revealed relatively large and immature cells of introduction of STI571, an ABL tyrosine kinase lymphoid appearance, which by surface marker inhibitor that shows very promising results in clinical analysis showed a null-cell phenotype. trials with BCR ± ABL-positive leukemias. STI571 is These approaches open the possibility to investigate considered by many clinicians to become a standard the molecular pathogenesis of ALCL caused by treatment of these leukemias in the near future. NPM ± ALK in an in vivo model. The contribution of Therefore, much eort in the next few years is focusing dierent signaling pathways, which have been char- on the development of analogous compounds for acterized only in cell lines thus far, to the oncogenicity inhibition of ALK. Whether such inhibitors will be of NPM ± ALK in vivo could be investigated by able to induce apoptosis of ALK-positive lymphoma employing dierent NPM ± ALK mutants in these cells and complete remissions in ALCL patients, as mouse models. In addition, the availability of knock- appears to be the case for STI571 and BCR ± ABL- out mice for certain signal transduction pathways now positive leukemias, remains to be seen. allows evaluation of the oncogenicity of NPM ± ALK Finally, immune-based therapeutic strategies could within a speci®c mouse knock-out background. This potentially be employed for ALK-positive ALCL. It powerful approach has been used very successfully for has been demonstrated that many patients with NPM ± studies of transformation mediated by the BCR ± ABL ALK-positive lymphomas have signi®cant levels of oncogene (Sexl et al., 2000; Li et al., 2001). Finally, circulating antibodies directed against the ALK portion these mice models are useful tools to evaluate potential of NPM ± ALK, suggesting that ALK fusions are quite NPM ± ALK inhibitors in animals. immunogenic (Pulford et al., 2000). Thus, several strategies for anti-ALK immune-based treatments of chemotherapy-resistant ALCL can be imagined. Clinical implications

ALK-positive lymphomas (`ALKomas') de®ne a dis- Conclusions tinct class of non-Hodgkin's lymphomas. The molecular characterization of this entity now allows the The identi®cation of activated ALK fusion proteins in development of molecularly targeted therapies. Since ALCL has revolutionized understanding of the genesis, the malignant clone is distinguishable based on the diagnosis, and classi®cation of these non-Hodgkin's expression of oncogenic ALK, the design of speci®c lymphomas. In addition, recent, ongoing studies are drugs targeted only against the malignant clone seems examining the role of ALK fusions in the genesis of achievable. Several strategies to speci®cally target in¯ammatory myo®broblastic tumors. In both of these ALK-positive tumor cells can be envisioned. For neoplasms, ALK promises to be an excellent target for example, NPM ± ALK-speci®c ribozymes could prevent directed therapeutic approaches for the future.

Oncogene Translocations involving ALK J Duyster et al 5635 Acknowledgments Institute (NCI) grants CA69129 and CA76301, and CORE This work was supported by a grant from the Wilhelm- grant CA21765 to SW Morris, and by the American- Sander Stiftung and Mildred-Scheel Stiftung to J Duyster, Lebanese Syrian Associated Charities (ALSAC), St Jude by SFB grant No 456 to J Duyster, by National Cancer Children's Research Hospital.

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