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2009;15:5609-5614. Published OnlineFirst September 8, 2009.Clin Cancer ResYael P. Moss, Andrew Wood and John M. MarisInhibition of ALK Signaling for Cancer Therapy

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Molecular Pathways

Inhibition of ALK Signaling for Cancer Therapy

Yael P. Moss,1,2 Andrew Wood,1,2 and John M. Maris1,2,3

Abstract Paradigm shifting advances in cancer can occur after discovering the key oncogenicdrivers of the malignant process, understanding their detailed molecular mechanisms,

and exploiting this transdisciplinary knowledge therapeutically. A variety of human

malignancies have anaplastic lymphoma kinase (ALK) translocations, amplifications,

or oncogenic mutations, including anaplastic large cell lymphoma, inflammatory myo-

fibroblastic tumors, nonsmall cell lung cancer, and neuroblastoma. This finding has

focused intense interest in inhibiting ALK signaling as an effective molecular therapy

against diseases with ALK-driven pathways. Recent progress in the elucidation of the

major canonical signaling pathways postulated to be activated by NPM-ALK signaling

has provided insight into which pathways may present a rational therapeutic approach.

The identification of the downstream effector pathways controlled by ALK should pave

the way for the rational design of ALK-inhibition therapies for the treatment of a subset

of human cancers that harbor ALK aberrations. (Clin Cancer Res 2009;15(18):560914)

Background

Anaplastic lymphoma kinase (ALK) is an orphan receptor ty-rosine kinase first identified as part of the t(2;5) chromosomaltranslocation associated with most anaplastic large cell lympho-mas (ALCL) and a subset of T-cell non-Hodgkins lymphomas(1). It has recently become clear that many human cancers ac-tivate ALK signaling by creating unique oncogenic fusions ofthe ALKgene at chromosomal band 2p23 with a variety of part-ners through chromosomal translocation events (2), resultingin the generation of oncogenic ALK fusion genes and their en-coded proteins. These oncogenic fusion proteins lead to consti-tutive activation of the ALK kinase domain and have beenidentified in various solid tumors, including inflammatorymyofibroblastic tumors (IMT; ref. 3), squamous cell carcinomas(4), and non-small cell lung cancers (NSCLC; ref. 5, 6). It hasalso recently been discovered that germline mutations in ALKare the major cause of hereditary neuroblastoma (7), and thatthese mutations can also be somatically acquired (710).

The extracellular region of ALK shows significant homology tothe leukocyte tyrosine kinase (11), which places ALK in the insu-lin receptor superfamily of RTKs. The ALK gene encodes a 1,620-

amino acid protein that undergoes posttranslational N-linkedglycosylation to a fully mature form weighing 220 kDa. ALKex-pression is restricted to the developing central and peripheralnervous system with a postulated role in participating in the reg-ulation of neuronal differentiation (12). The absence of pub-lished structural studies of ALK extracellular and intracellulardomains, and the conflicting literature over whether pleiotro-phin (13, 14) and/or midkine (15) are ALK ligands, emphasizesthat much remains to be learned about ALK structure, function,and signaling. Recently it has become clear thatALKisone ofthe

few oncogenes activated in both hematopoietic and nonhemato-poietic malignancies. Although constitutive ALK signaling hasbeen shown in these contexts to induce cell transformation in vi-tro and in vivo by controlling key cellular processes such as cell-cycle progression, survival, cell migration, and cell shaping, thecanonical signaling pathways and cell-type specificities of signal-ing remain poorly defined. Regardless, it is now clear that ALKrepresents a tractable target for innovative therapies on the basisof selective inhibition of its tyrosine kinase activity. This reviewfocuses on the normal and cancer-related biology of ALK and thepreclinical proof-of-principle for ALK as a therapeutic target.

Role of ALK in Cancer

A variety of mechanisms that lead to aberrant ALK signalingin a variety of human cancers have been characterized, andthese include translocations or structural rearrangements, ALKgene amplification, mutations, and overexpression (Fig. 1). Im-portantly, missense germ-line mutations in the tyrosine kinasedomain have been described in patients with hereditary neuro-blastoma, with acquired somatic mutations seen in the morecommon sporadic form of the disease.

ALK Fusion Proteins-Translocations

Translocations are the most common known cause of ge-nomic ALK aberration. Recent data suggest that deregulation

Authors' Affiliations:1Division of Oncology and Center for ChildhoodCancer

Research, Children's Hospital of Philadelphia, 2Department of Pediatrics,

University of Pennsylvania School of Medicine, and 3Abramson Family

Cancer Research Institute, University of Pennsylvania School of Medicine,

Philadelphia, Pennsylvania

Received 5/14/09; revised 5/22/09; accepted 5/28/09; published OnlineFirst

9/8/09.

Grant support: National Institutes of Health (NIH) grants K08-111733 (Y.P.

Moss), the Fulbright Foundation (A. Wood), R01-CA78454 (J.M. Maris),

R01-CA124709 (J.M. Maris), and the Giulio D'Angio Endowed Chair (J.M.

Maris).

Requests for reprints: John M. Maris, Children's Hospital, Philadelphia, PA

19104-4318. Phone: 215-590-5242; Fax: 215-590-3770; E-mail: [email protected].

F2009 American Association for Cancer Research.

doi:10.1158/1078-0432.CCR-08-2762

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of several genes (Fra 2 on 2p23, HLH protein Id2 on 2p25, andthe CSF1 receptor on 5q33.1) located near the ALCL transloca-

tion breakpoint occurs before the formation of translocationevents, and that aberrant transcriptional activity of certain ge-nomic regions is linked to their propensity to undergo chromo-somal translocations (16). In physiological ALK signaling,ligand-induced homo-dimerization of the extracellular do-mains is hypothesized to bring the tyrosine kinase domainsinto sufficient proximity to enact trans-phosphorylation andkinase activity. By contrast, translocations resulting in patho-genic fusion partners provide dimerization domains that areligand independent, leading to unregulated constitutive kinaseactivity and malignant transformation.

Approximately 70 to 80% of ALK-positive ALCL express thenucleophosmin-ALK (NPM-ALK) fusion protein derived from

the t(2;5)(p23;q35) translocation, and about the same fre-quency of ALCLs stain positive for ALK by immunohistochem-

istry (17, 18). Within the truncated nucleophosmin protein theessential domain required for transformation is a coiled-coiloligomerization domain. Site-directed mutagenesis of the di-merization domain abrogates NPM-ALK-transforming activitysuggesting that proximity of the ALK kinase domains is a pre-requisite to facilitate trans-phosphorylation of the partnerkinase (19). The truncated NPM also encodes a nucleolar local-ization domain that explains why in contrast to other ALK fu-sions, NPM-ALK can be detected in the cytoplasm, nucleus, andnucleolus (2), whereas most fusion products are largely cyto-plasmic. The remaining 20% of translocations fuse ALKto otherpartners that commonly encode coiled-coil oligomerization do-mains such as Tyrosine Receptor Kinase-fused gene (TFG),

Fig. 1. A, representation of ALKaberrations in human cancers and

major signaling pathways activatedthrough NPM-ALK. ALCL and IMTshave unique and shared fusionpartners, but no overlap with NSCLC.Mutations and amplification are uniqueto neuroblastoma but rarely co-occur;translocations have not been detectedin neuroblastoma specimens. Cancersdysregulate ALK via multiplemechanisms. B, ligand-induceddimerization of the extracellulardomain of ALK monomers leads totrans-phosphorylation of the tyrosinekinase (TK) domain and physiologicalALK signaling. C, ligand-independentsignaling is induced by activatingmutations within the TK domain,or amplification of wild-type ALK

mediating overexpression,spontaneous dimerization, andtransactivation of the TK domain.D, fusion partners facilitateapproximation of truncated ALKcontaining the TK domain throughcoiled-coil domains or subcellularlocalization. E, constitutive ALKsignaling induces transformation viacanonical signaling networks. SignalTransducers and Activation ofTranscription protein 3 may couplecell growth to increased oxidativephosphorylation, throughphosphorylation of tyrosine and serineresidues, respectively. In ALCL, ALKsignaling cooperates with othergenetic aberrations. AKT stabilizesthe GLI1 protein that is upregulatedvia amplification of the SonicHedgehog locus.

5610Clin Cancer Res 2009;15(18) September 15, 2009 www.aacrjournals.org

Molecular Pathways


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Tropomyosin 3 (TPM3), and Tropomyosin 4 (TPM4). There arealso several nonnuclear ALK fusion chimeras that induce transfor-mation in vitro. The clathrin heavy chain-like gene (CLTCL), forexample, encodes a clathrin family member that coalesces to pro-duce cytoplasmic vesicles of CTLC-ALK bringing the ALK tyrosinekinase domains into close proximity (20). Although almost all

ALK fusions retain the same portion of ALK because of transloca-tion events that result in loss of the entire extracellular portion

of normal ALK as well as the transmembrane segment, they showslight differences in pathway activation, likely because of differ-ences in their subcellular compartmentalization and/or cell-type.NPM-ALK transgenic mouse models have shown T-cell trans-formation as well as B-cell lymphomas (21) and may be usefulin the evaluation of new therapeutic agents for these diseases.

The deregulated expression of full length ALK and ALK fu-sions has recently been observed in several nonlymphoid neo-plasms. Interest in pharmacological inhibition of ALK has mostrecently been spurred by the discovery of transforming echino-derm microtubule associated protein like 4-anaplastic lympho-ma kinase (EML4-ALK) fusions in a subset of NSCLCs (5), anda recent report of a novel fusion transcript, kinesin family mem-

ber 5B-anaplastic lymphoma kinase (KIF5B-ALK), in this dis-ease (22). Dimerization is enabled as the truncated EML4protein retains a coiled-coil oligomerization domain in a man-ner similar to NPM-ALK. Epidemiologic characterization ofEML4-ALK translocations is ongoing but it seems to be a rareaberration, most common in Asian nonsmokers or light-smokers

with the adenocarcinoma subtype of NSCLC, forming a distinctsubgroup from patients harboring EGFR, KRAS, or NKX2-1 aber-rations (2326). The initial frequency of EML4-ALK fusion tran-scripts was approximately 6.7% in Japanese NSCLC tumorsamples (5), but subsequent studies showed lower frequenciesof 3% or less (24, 25, 27). Direct demonstration of EML4-ALKas a dominant oncogenic driver in NSCLC includes transforma-tion of mouse 3T3fibroblastic cells with forcedoverexpressionofhuman EML4-ALK and de novo lung adenocarcinoma formationin mice engineered to express EML4-ALK targeted to the lungalveolar epithelial compartment (28).

IMT is a rare tumor of mesenchymal origin that arises in mul-tiple tissues, possesses metastatic potential (29), and is onlyminimally responsive to conventional chemotherapy or radia-tion therapy. IMT is associated with various ALKtranslocationsthat overlap with those found in ALCL, including TPM3 andTPM4 (30), CARS (31), and CLTC (32), but not to date withfusion partners seen in NSCLC. Immunohistochemical ALK re-activity occurs in 56% of cases and is restricted to the cytoplasmconsistent with the observation that NPM-ALK translocationsdo not occur in this disease. A subset of esophageal cancers

show the TPM4-ALK fusion protein, but frequency and signifi-cance of this has not been fully defined (33).

Mutations and Genomic Amplification

Expression of ALK has been reported in a large fraction ofhuman-derived neuroblastoma cell lines (34), and our groupand others had previously identified ALK as a candidate neuro-blastoma oncogene through somatically acquired amplificationof the genomic locus (35, 36). Recently, both germline (7) andsomatic (710) mutations that activate ALK have been discov-ered in neuroblastoma, an often lethal embryonal malignancythat contributes 15% to the overall childhood cancer mortality

rate (37). Heritable gain-of-function mutations in ALKare thecause of most hereditary neuroblastoma cases; one of the fewpediatric tumors to be initiated by gain-of-function mutations.

To date, heritable mutations have been restricted to the kinasedomain of ALK, and it is presumed, but not yet proven, thatthe second hit is somatic gain or amplification of the mutant al-lele, similar to what is seen at the METlocus in hereditary renalpapillary cancer (38). Although the number of families studied

remains relatively small, it is apparent that theR1275Q mutationis most common, and it seems that disease penetrance maybe attributable to mutation type as rarer mutations, such asG1128A seen in one large pedigree, was associated with verylow penetrance compared with the near complete penetranceseen with the R1275Q mutation. Both mutations fall withinthe kinase activation loop in a region strongly associated withactivating mutations seen in other oncogenic kinases (7). Thisfact compounds the already controversial topic of the role forgenetic screening in this disease. Because there are noninvasivemethods to screen for neuroblastoma (urinary catecholaminemetabolites have relatively high sensitivity and specificity fordetecting disease), we currently recommend genetic testing

of the proband in any case in which there is a first or seconddegree relative with neuroblastoma, or the proband has multifo-cal disease consistent with hereditary predisposition. Becauseneuroblastoma often occurs in families with other young chil-dren or plans for future children, there is often parental interestin testing even when there is no objective evidence for hereditarypredisposition. As we define the frequency of occult germlinemutations and further understand the role of ALK in the initia-tion of neuroblastomas without a family history, screeningrecommendations can be solidified.

ALKactivation by mutation and/or amplification is function-ally relevant in models of high-risk neuroblastoma, and thusmight offer a tractable therapeutic target. We and others haveshown thatALKis expressedin themajorityof human neuroblas-toma-derived cell lines, and thatALKexpression is significantlyhigher in neuroblastoma cells harboring ALK mutations (39).

Analysis of protein lysates from these lines showed constitutivephosphorylation in each of the cell lines harboring mutations,

with weak phosphostaining in a smaller subset of wild-type celllines, suggesting that ALK activation can occur by mechanismsother than mutation within the kinase domain. Transient siRNAknockdown directed against ALK in neuroblastoma cell linesshowed significant inhibition of growth restricted to cells withevidence for ALK activation. Baf3 cells expressing the two mostcommon mutations showed cytotoxicity to NVP-TAE684, asmall molecular competitive inhibitor of ATP binding inthe ALK kinase domain (40). NVP-TAE684 has been shown

to block the growth of ALCL-derived and ALK-dependent celllines with IC50 values between 2 and 10 nM; however, due toits toxicity profile, it is now only used as a tool compound(40). More recently, pharmacologic inhibition with a highly spe-cific small molecule inhibitor of ALK currently in phase 1 clinicaltrials (PF-02341066, see Clinical Translational Advances)showed potent antitumor activity in preclinical models ofneuroblastoma both in vitro and in vivo, in a mutation-specificmanner. Although these data are still emerging, it is somewhatclear that ALK phosphorylation status is a good biomarker forsensitivity to pharmacologic inhibition, but there seem to bemutation-specific differences in sensitivity. Solving the crystalstructure of the ALK kinase domain, and cocrystallization with


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pharmacologic inhibitors, will be necessary to understand ifsome mutations confer resistance, and to understand how resis-tance mutations may arise under selective pressure. In addition,defining the downstream signaling pathways that mediate theef-fects of ALK activity in the context of a neuroblastic cell will alsobe critical, especially for the development of rational combina-tion therapies. Work is ongoing to characterize the full spectrumof germline and somatic DNA alterations leading to ALKactiva-

tion to develop strategies for future clinical trials on the basis ofinhibiting ALK-mediated signaling in neuroblastoma.

Expression

ALK expression is of uncertain pathogenic significance inseveral human cancers. The detection of ALK mRNA in diversehuman tumor-derived cell lines (41), as well as the detection ofhigher ALK expression in tumor compared with neighbor-ing normal tissue (15), likely reflects physiological expressionrather than seminal pathogenic events (41). ALK has previouslybeen shown to be upregulated and functionally relevant in glio-blastoma, and recent work has shown that ribozyme-mediated

knockdown of ALK results in abolishment of tumor growth in axenograft model (42). This offers potentially novel therapeuticoptions for an otherwise lethal brain tumor. Immunohistochem-ical detection of ALK protein overexpression is pathognomonicfor ALCL, and recent work suggests that ALK immunohistochem-istry may have utility as a screening tool or surrogate markerfor EML4-ALK fusion gene-positive NSCLC tumors (43). Cur-rently work is focused on developing reliable techniques forphospho-ALK staining in routine clinical specimens, and thismay be critically important to properly evaluate future ALKinhibitor clinical trials.

Differential Activation of Downstream Signaling

PathwaysThe critical pathways involved in transformation because of

dysregulated ALK signaling are best characterized by transloca-tions that juxtapose ALK to dimerization partners (Fig. 1; ref. 2).

The constitutive activation of ALK fusion proteins leads to cel-lular transformation through a complex signaling network.

Among the potential combinations of proteins phosphorylatedby ALK kinase activity, it has been postulated that the most im-portant effects involve activation of STAT3 (44, 45), AKT/PI3K(46), and RAS/ERK (47) pathways, which control cell prolifer-ation, survival, and cell cycling. These pathways are typicallyupregulated in transformed cell lines (48), and pharmacologi-cal inhibition of ALK leading to apoptosis abrogates phosphor-

ylation of these key signal-transducing proteins in ALCL models(49, 50). Inhibition of downstream signal transduction pro-teins, such as STAT3, has been shown to prevent NPM-ALK in-duced transformation in vivo (45). Recent work has shown thatthe sonic hedgehog signaling pathway (SHH/GLI1) is also acti-

vated in ALK-positive ALCLs (51). SHH/GLI1 activation is theresult of SHH gene amplification and is further mediated byNPM-ALK through activation of PI3K/AKT and stabilizationof GLI1 protein (51). Inhibition of this pathway induces apo-ptosis and cell cycle arrest, suggesting that inhibition of SHH/GLI1 signaling in combination with blocking NPM-ALK and/orthe PI3K/AKT pathway may present a rational therapeutic ap-proach in ALK+ ALCL.

Different ALK aberrations produce diverse pathogenic signal-ing anomalies through a combination of differentially activatingcommon signal transduction pathways and unique pathogenicmechanisms. Different ALK fusion proteins transfected intoNIH3T3 cells produced clones with differential signaling andphenotypic differences beyond those that could be explainedby the variable kinase activity of each fusion. TPM3-ALK cellsshowed the most AKT phosphorylation and migratory capacity

in vitro, but in vivo tumorigenicity was less than that observedin NPM-ALK cells. In contrast, ATIC phosphorylation enhancesenzymatic activity of NPM-ALK (52), and ATIC-ALK cells causeSTAT3 phosphorylation with only modest invasion and in vivotumorigenicity (48). TPM3-ALK invasive capacity may partly bemediated by a unique pathogenic mechanism. TPM interacts

with endogenous tropomyosin in the cytoskeleton, which is hy-pothesized to alter cytoskeletal organization and led to greatermotility and metastatic potential (29). It has been postulatedthat the STAT family is unlikely to play a key role of the patho-genesis of EML4-ALK (53), even though STAT3 anti-senseoligonucleotides suppress the in vivo transforming activity ofNPM-ALK (45). Tissue context and signaling differences may

explain why some ALCL and IMT share some fusion partners,but display no overlap with NSCLC. Additional signaling andphenotypic variation will likely be discovered when the fullspectrum of mutated ALK and pathogenically overexpressed

wild-type (via genomic amplification) prot eins are studied.These signaling variations could arise because of multiple me-chanisms including: variable subcellular localizations of activated

ALK, altered sequences of tyrosine auto-phosphorylation, alteredkinase substrate specificity, tissue context, autocrine or paracrineligand effects, and by breakpoints disrupting the original loci in

which truncated genes are translocated from.

Clinical Translational Advances

Small molecule tyrosine kinase inhibitors. Kinases are criticalcomponents of cellular signal transduction cascades, and arekey effectors of cell proliferation and differentiation. Therenow exists clear precedent in clinical oncology that robust anti-tumor activity can be obtained with inhibitors directed towardoncogenic tyrosine kinases that are genetically dysregulated (54,55), and these lessons are readily applicable to ALK kinase in-hibition. Imatinib showed that off-target kinase inhibitioncould be rationally exploited because of its efficacy in Kit+ gastro-intestinal stromal tumors (56). This is significant, as the first ALKinhibitor to enter phase 1 clinical trials, PF-02341066, was ini-tially designed to inhibit c-Met, but was also found to have sig-nificant activity against ALK (50). This orally bioavailable small

molecule inhibitor caused complete regression of NPM-ALKxenografts at pharmacologically relevant doses, with a strongcorrelation between antitumor response and abrogation of phos-phorylation of ALK (50). PF-02341066 is currently the onlyavailable ALK small-molecule inhibitor in clinical trials; how-ever, recognition of the variety of malignancies in which ALKplays a causative role has prompted broad developmental effortsin this area. Point mutations in the kinase domain of other tyro-sine kinases such as BCR-ABL, KIT, and EGFR impair drug bind-ing as the major mechanism of acquired resistance to kinaseinhibitors. To begin to address this, investigators have generatedseveral ALK mutants in the kinase domain and evaluated theirkinaseactivity andsensitivity to inhibition by two ALKinhibitors


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developed from different chemical series (57). Binding of theseinhibitors to wild-type and mutant ALK was analyzed usinghomology models, and showed that some of the mutants are re-sistant to select small molecule inhibitors. It remains to be seen

whether newer inhibitors designed with greater specificity againstALK will eventually prove superior to multikinase inhibitor.

Future Developments

ALK is one of the few examples of a receptor tyrosine kinaseimplicated in the oncogenesis of both hematopoietic and non-hematopoietic malignancies. Although the importance ofautocrine-paracrine growth loops involving normal ALK in tu-morigenesis is not yet fully elucidated, there are compelling da-ta to support the hypothesis that tumors with ALK aberrationsexhibit oncogene addiction, with inhibition of aberrant ALKsignaling resulting in marked antitumor efficacy in preclinicalmodels. Additional studies are required to determine whether tu-mors that express full-length ALK that is activated by its ligandexhibit the same or at least partial dependence upon ALKgrowth signals.

There is strong rationale and significant enthusiasm towardALK inhibition as a targeted therapy against tumors harboringoncogenic fusions or activating mutations, and the results ofthe first clinical trial testing the PF-02341066 dual c-Met/ALKinhibitor has shown clinical activity in highly refractory patients

with tumors harboring ALK translocations, leading to ongoingefforts to select patients for these trials based on ALK transloca-tion status. Defining the epidemiology of ALK aberrations ineach cancer that harbors ALK pathway activation seems essential

to properly design and interpret clinical trials of ALK inhibitors.Furthermore, as potency will likely be variable on the basis of themechanism for pathway activation, and the emergence of resis-tance mechanisms almost assured, co-development of small mo-lecular and monoclonal antibody inhibitors of ALK activationseems prudent. A pediatric trial of PF-02341066 will begin en-rollment of subjects in 2009, and plans are currently being madefor how to properly integrate ALK inhibition therapy into front-

line chemotherapeutic regimens. If the preclinical models do in-deed predict what will be observed in the clinic, it is clear that forcancers that can usurp ALK signaling as a mechanism of oncoge-nicity, ALK pathway status must be assayed for at the time ofdiagnosis or relapse.

Cautious extrapolation from preclinical models and inhibitor-kinase co-crystallization studies will help us to define biomarkerspredictive of therapeutic response. Besides the use of selective

ALK inhibitors, it may be crucial to develop strategies to inhibitmultiple targets involved in the ALK-signaling pathway. siRNAscreens of the druggable genome in combination with ALK inhi-bitors, and preclinical testing for synergy and antagonism withexisting chemotherapy backbones will be important to maxi-

mize efficacy. Lastly, the development of monoclonal antibodiesagainst ALK should be pursued, a precedent set by the develop-ment of monoclonal antibodies against members of the EGFRfamily, and an approach that may prove to be particularly usefulfor tumors with ALK gene amplification.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

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