TheNeuroblastomaALK ( I 1250 T ) Mutation Is a Kinase-Dead RTK In Vitro and In Vivo 1 , 2

Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations—ALK—identified in the germ line DNA of one patient. Our analyses were performed in cell culture–based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALK mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALK and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALK is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALK mutant. Identical results were obtained when the ALK mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALK mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-offunction status, with significant implications for patients reported to carry this particular ALK mutation. Translational Oncology (2011) 4, 258–265 Address all correspondence to: Prof. Bengt Hallberg or Prof. Ruth Palmer, Department of Molecular Biology, Building 6L, Umeå University, Umeå S-901 87, Sweden. E-mail: Bengt.Hallberg@molbiol.umu.se, Ruth.Palmer@ucmp.umu.se This work has been supported by grants from the Swedish Cancer Society (08-0597 to B.H.), the Children’s Cancer Foundation (08/084 to B.H. and 08/074 to R.H.P.), the Swedish Research Council (621-2003-3399 to R.H.P.), Lions Cancer Society (to B.H. and R.H.P.), Umeå and the Association for International Cancer Research (080177 to R.H.P.). S.K. is a Children’s Cancer Foundation fellow (NBCNSPDHEL09/ 002). R.H.P. is a Swedish Cancer Foundation Research Fellow. This article refers to supplementary materials, which are designated by Figures W1 to W3 and are available online at www.transonc.com. Joint first authors. Received 22 March 2011; Revised 22 March 2011; Accepted 24 March 2011 Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1944-7124/11/$25.00 DOI 10.1593/tlo.11139 Introduction During 2008, anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) was identified as a familial predisposition gene for the development of neuroblastoma [1]. This study was further supported by four independent reports of additional activating ALK mutations in both familial and somatic neuroblastomas [2–5]. Neuroblastoma is a neural crest–derived embryonal tumor of the postganglionic sympathetic nervous system. Further, it is the most common single solid tumor of childhood with the worst prognosis, constituting almost 6% of diagnosed tumors and more than 9% of all deaths [6]. The origin of these tumors remains unknown in most cases, although a number of familial cases have recently been associated with mutations of the ALK gene [1,5]. Neuroblastomas show heterogeneous biologic and clinical features and, whereas a subset may undergo spontaneous differentiation or regression with little or no therapy, the majorities are difficult to cure with current modalities. The Translational Oncology Vol. 4, No. 4, 2011 ALK Mutation in NB Tumor Schönherr et al. 259 ALK RTK was first described in the mid-1990s, and aberrant ALK protein activity is now implicated in a range of nonhematopoietic, hematopoietic, as well as neuroendocrine tumors (for review, see Palmer et al. [7]). At this point, there are no clinically approved treatments of aberrant regulated or oncogenic ALK expression, although recent studies provide an optimistic view of Crizotinib, a small ALK and c-Met inhibitor in ALK-positive non–small cell lung cancer and inflammatory myofibroblastic tumor [8–10]. One possible positive offshoot is the potential use of Crizotinib to treat neuroblastoma patients [7]. In this study, we have investigated the described ALK mutation discovered in a neuroblastoma patient [1]. This mutation had not previously been described in either the SNP database (dbsSNP; http://www.ncbi.nlm.nih.gov/projects/SNP/) or in the somatic mutation database (COSMIC; http://www.sanger.ac.uk/genetics/CGP/ cosmic) [1]. The ALK mutation was present in the matched germ line DNA, raising the possibility of hereditary predisposition [1]. The original in silico analysis predicted ALK to be an activating mutation in ALK [1]. During 2010, the crystal structure of the ALK kinase domain was described by two groups [11,12], in which the ALK catalytic loop mutation was described as promoting oncogenesis by altering the substrate binding to become a gain-of-function mutation [11], further reinforcing the notion that ALK is an activated mutant. However, to our own surprise and in contrast to previous predictions, we clearly observe that rather than being a gain-of-function mutation, the ALK mutant is actually a kinase-dead RTK. Materials and Methods Generation of Human and Mouse ALK Mutant Constructs in Cells and Drosophila Construction of the mouse 3761 T→C point mutation, corresponding to the mouse I1254T mutation, and the human 3749 T→C, corresponding to the human I1250T mutation was performed using QuickChange Site-Directedmutagenesis kit (Stratagene, [CedarCreek, TX] according to the manufacturer’s instructions) with the following primers: mouse 5′-CACTTTATCCACCGGGATACTGCTGCTAGAAACTG-3′ and 5′-CAGTTTCTAGCAGCAGTATCGGCGTGGATAAAGTG-3′ and human 5′-ACCACTTCATCCACCGAGACACTGCTGCCAGAA-3′ and 5′-TTCTGGCAGCAGTGTCTCGGTGGATGAAGTGGT-3′. All constructs were confirmed by sequencing from both directions. The mouse ALK fragment was ligated into the pTTP vector, described in Schonherr et al. [13], resulting in the pTTPmALK I1254T plasmid. Human pcDNA wild-type and F1174S ALK have been described [14]. The human ALKI1250T fragment was ligated into full-length human ALK in both pTTPhALK and pcDNA3(hALK) [14]. Antibodies and Inhibitors The following antibodies were used: anti–pan-ERK (1:5000) was purchased from BD Transduction Laboratories (Franklin Lakes, NJ), and the anti–p-ERK was fromCell Signaling Technology (Danvers, MA). The activating monoclonal antibodies 46 and 31 (mAb46 and 31) have been described previously [14,15]. Monoclonal Ab no. 153 was produced in the laboratory against the extracellular domain of ALK in similar manners as described [15]. The anti–phosphotyrosine antibody 4G10 was from Upstate Biotech (Lake Placid, NY). Organelle marker antibodies, anti– GRP78/BiP rabbit antibody (ab21685) and anti–GM130 rabbit antibody (ab52649), were obtained from Abcam (Cambridge, United Kingdom). Cy2-labeled goat antimouse IgG and Cy3-labeled goat antirabbit IgG were from GE Healthcare (Uppsala, Sweden). The horseradish peroxidase–coupled secondary antibodies goat antirabbit IgG and goat antimouse IgG were from Thermo Scientific (Waltham, MA). The ALK-specific inhibitor NVP-TAE684 has been described previously [13,16]. Cell Lysis, Immunoprecipitation, and Western Blot Analysis Briefly, both mouse and human PC12ALK cells were induced with doxycycline and serum starved for 20 hours. PC12mALK cells were additionally stimulated with 1 μg/ml of the activating mAb46 for 30 minutes [13,15,17]. Precleared cell lysates were analyzed on SDS/ PAGE, followed by immunoblot analysis with the indicated antibodies. ALK downstream activation was detected by p-ERK, and pan-ERKwas used to show equal loading. ALK-phosphorylation was detected by the 4G10 antibody. Cell lysis, immunoprecipitation, and immunoblot analysis were performed according to the protocols described in Schonherr et al. [13]. Neurite Outgrowth Assay Both PC12mALK and PC12mALK cells were seeded sparsely in six-well plates, and ALK expression was induced by doxycycline. PC12mALK cells were stimulated with 1 μg/ml mAb46. Quantification of neurite outgrowth in the cells was carried out as described [15]. Experiments were performed in triplicates, and each sample within an experiment was performed in duplicate. For human ALK, 2 × 10 PC12 cells were transfected by electroporation in an Amaxa electroporator (Amaxa, Cologne, Germany) using 0.8 μg of pcDNA3-hALK and 0.8 or 1.6 μg of pcDNA3-hALK and 0.5 μg of pcEGFPN1 (Clontech, Mountain View, CA) and 100 μl of Ingenio electroporation solution (Mirrus Bio LCC, Madison, WI). After transfection, cells were transferred to Dulbecco modified Eagle medium (DMEM) supplemented with 7% horse serum and 3% fetal bovine serum, thereafter seeded into 24-well plates together with 1 μg/ml mAb31. Two days after transfection, the fraction of GFP-positive and neuritecarrying cells versus GFP-positive cells was estimated under a Zeiss Axiovert 40 CFL microscope (Carl Zeiss, Stockholm, Sweden). To be judged as a neurite-carrying cell, the neurites of the cell had to reach at least twice the length of the diameter of a normal cell body. Transformation Assay Low-passage number NIH 3T3 cells (ATCC, Manassas, VA) were transfected with Lipofectamine 2000 according to the manufacturer’s protocol. Briefly, 4.5 × 10 cells, seeded the day before into collagencoated 12-well plates, were transfected for 6 hours with 0.55 μg of pcDNA3 containing hALK, hALK, or hALK DNA and 1.4 μl of Lipofectamine 2000 in 0.3 ml of Opti-MEM. Twenty-four hours after transfection, three fifths of the cells from each well were transferred to wells in 12-well plates and kept in DMEM (10% fetal calf serum [FCS] and 0.5 mg/ml G418) until the cells reached confluence. Thereafter, cells were kept in DMEM (5% FCS and 0.25 mg/ml G418) for another 10 days. Cell Culture and Immunofluorescence CLB-GE neuroblastoma cell line was grown as described [5]. HEK293 cells were grown in DMEM containing 10% heat-inactivated FCS on