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. 2008 Oct 16;455(7215):930-5.
doi: 10.1038/nature07261. Epub 2008 Aug 24.

Identification of ALK as a major familial neuroblastoma predisposition gene

Yaël P Mossé  1 , Marci Laudenslager, Luca Longo, Kristina A Cole, Andrew Wood, Edward F Attiyeh, Michael J Laquaglia, Rachel Sennett, Jill E Lynch, Patrizia Perri, Geneviève Laureys, Frank Speleman, Cecilia Kim, Cuiping Hou, Hakon Hakonarson, Ali Torkamani, Nicholas J Schork, Garrett M Brodeur, Gian P Tonini, Eric Rappaport, Marcella Devoto, John M Maris
Affiliations

Identification of ALK as a major familial neuroblastoma predisposition gene

Yaël P Mossé et al. Nature. .

Abstract

Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23-24 using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194 high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability, to be oncogenic drivers. Mutations resulted in constitutive phosphorylation, and targeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in all cell lines harbouring mutant or amplified ALK, as well as in two out of six wild-type cell lines for ALK. Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.

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Figures

Figure 1
Figure 1. Eight neuroblastoma pedigrees with ALK mutations
All family members with DNA available for genotyping indicated with either wild type (wt) for ALK, or with mutation in the ALK tyrosine kinase domain (R1192P, R1275Q, G1128A). Individuals affected by neuroblastoma indicated by filled symbol.
Figure 2
Figure 2. Germline and somatic ALK mutations
a. Schematic indicating protein structure of ALK, with mutations discovered in constitutional DNAs of familial cases (germline) and primary tumors from sporadic cases (somatic) indicated. All but one sequence alteration mapped to the tyrosine kinase domain (D1091N was just N-terminal and is not indicated here). Of the three germline mutations discovered, only the R1275Q was found in the tumor DNA samples. Conversely, the I1250T mutation discovered in the tumor set was also present in the matched germline DNA of that patient, while all of the other mutations studied here were somatically acquired. b. Homology model of wild-type ALK with each major subdomain indicated, . c. ALK mutations mapped onto homology model (orientation different to show all mutations) with colors indicating subdomain in which the mutation resides (e.g. the R1275Q mutation falls within the activation segment, indicated in green).
Figure 3
Figure 3. Representative ALK copy number alterations in three neuroblastoma primary tumors
Hybridization intensity reflecting copy number for all SNPs along chromosome 2p in three primary tumors from patients with sporadically occurring disease is shown, represented on a logarithmic scale. MYCN amplification is present in all tumors. a. Regional gain (trisomy) of chromosome 2p, including the ALK locus. b. Focal gain of the ALK locus. c. Focal amplification of the ALK locus. d and e. Complex rearrangement of the 2p locus, showing various focal amplicons, including MYCN and ALK.
Figure 4
Figure 4. ALK is highly expressed and the kinase is phosphorylated in neuroblastoma cell lines harboring activating mutations
a. Relative ALK expression of neuroblastoma cell lines and fetal brain is shown and was determined using the 2-ΔΔCt method. Statistical significance was determined by unpaired T-test. b. Immunoblots showing differential ALK expression in neuroblastoma cell lines with phosphorylation of the tyrosine 1604 codon restricted to cell lines with mutations (the wild-type lines NBEC1 and NB1771 show faint phosphostaining).
Figure 5
Figure 5. Inhibition of growth in ALK mutated or amplified neuroblastoma cell lines
Substrate adherent cellular growth was monitored and recorded every 30 minutes for at least 120 hours in ten neuroblastoma cell lines that were untreated, transfected with siRNAs against ALK, GAPDH (negative control), a non-targeting control (NTC, negative control) or the polo-like kinase gene (PLK1; positive control). The x-axis is time in hours after transfection, and differs based on the growth kinetics of each cell line. The y-axis is percent growth normalized to the siRNA against GAPDH. The normalized average percent growth is shown for siRNAs against GAPDH (red) and ALK (blue). In each experiment the siRNA NTC did not differ significantly from the siRNA against GAPDH, and in each case the siRNA against PLK1 showed profound growth inhibition, but only the siRNA against GAPDH and ALK are shown here for ease of visualization. Each experiment was plated in triplicate, and each experiment was repeated at least once with concordant results (data not shown). The replicates for each siRNA curve showed little variation with standard deviations <5% in all lines (except the siRNA against GAPDH in SKNDZ and NB1643 which were <10%). The cell lines, their mutation status and percent ALK mRNA knockdown at 48 hours are as follows: a. KELLY (F1174L) 78% mRNA knockdown (kd); b. SKNSH (F1174L), 21% mRNA kd; c. NB1643 (R1275Q), 48% mRNA kd; d. IMR5 (AMPLIFIED), 60% mRNA kd; e. NBEBC1 (WT), 74% mRNA kd; f. NB1771 (WT), 68% mRNA kd; g. SKNAS (WT), ALK not expressed at detectable levels; h. SKNDZ (WT), 41% mRNA kd; i. SKNBE2C (WT), 62% mRNA kd; j. NGP (WT), 86% mRNA kd. k. Summary of percentage growth inhibition with ALK siRNA knockdown by ALK mutational and allelic status; l. Immunoblot showing ALK protein knockdown at 24, 48 and 72 hours following siRNA transfection in the representative neuroblastoma cell lines KELLY (F1174L mutation and showing growth inhibition) and SKNDZ (wild-type ALK sequence and no growth inhibition).
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