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Activating mutations in ALK provide a therapeutic target in neuroblastoma

Abstract

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer1. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal2,3. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

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Figure 1: ALK mutant alleles F1174L and R1275Q are activating in Ba/F3 cells and are sensitive to pharmacological inhibition.
Figure 2: Neuroblastoma cell lines harbouring the F1174L ALK mutation, but not the R1275Q ALK mutation, are dependent on the altered protein for growth and survival.
Figure 3: The constitutively activated F1174L ALK protein has a higher turnover rate than wild-type ALK in neuroblastoma cell lines.

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Gene Expression Omnibus

Data deposits

Microarray data have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) public database. The accession numbers for the SNP array analyses are GSM206563 and GSM206564.

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Acknowledgements

We thank J. Elechko and L. Moreau for technical assistance, A. Azarova for help with cell culture and cell growth assays, Q. Jiang and X. Cui for assistance during ALK small-molecule inhibitor development, and A. Kung for Ba/F3 cells and retroviral vectors. We acknowledge the Children’s Oncology Group for provision of neuroblastoma tumour and matched normal DNAs, and tumour touch prep slides. We thank J. Maris for neuroblastoma cell lines. We thank Abbott Molecular International for the Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe. This work was supported by grants from the National Institutes of Health (R.E.G.), the Friends for Life Neuroblastoma Fund (R.E.G.), the Children’s Oncology Group (R.E.G.), Alex’s Lemonade Stand Foundation (M.H. and M.M.), NCI CA69129 (L.X. and S.W.M.), Cancer Center Core grant CA21765 (T.R.W., L.X. and S.W.M.), the American Lebanese Syrian Associated Charities and St. Jude Children’s Research Hospital (T.R.W., L.X. and S.W.M.).

Author Contributions R.E.G., M.M. and A.T.L. designed the experiments and wrote the manuscript. M.H., H.G. and M.M. performed the DNA sequencing and analysis. T.S., S.F., W.L., Y.A., H.G. and R.E.G. carried out the functional analyses. J.Z., W.Z. and N.S.G. performed the homology modelling and synthesis of TAE684. W.B.L. and P.M. performed the statistical analysis. S.Z., V.E.G. and T.R.W. were involved with the design of ALK inhibitors. L.X. and S.W.M. assisted with experimental design, provided reagents and advice on ALK inhibitors. M.M., L.D. and D.G.G. provided advice on the manuscript.

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Correspondence to Matthew Meyerson or A. Thomas Look.

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George, R., Sanda, T., Hanna, M. et al. Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature 455, 975–978 (2008). https://doi.org/10.1038/nature07397

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