Oncogenic mutations of ALK kinase in neuroblastoma

Abstract

Neuroblastoma in advanced stages is one of the most intractable paediatric cancers, even with recent therapeutic advances1. Neuroblastoma harbours a variety of genetic changes, including a high frequency of MYCN amplification, loss of heterozygosity at 1p36 and 11q, and gain of genetic material from 17q, all of which have been implicated in the pathogenesis of neuroblastoma2,3,4,5. However, the scarcity of reliable molecular targets has hampered the development of effective therapeutic agents targeting neuroblastoma. Here we show that the anaplastic lymphoma kinase (ALK), originally identified as a fusion kinase in a subtype of non-Hodgkin’s lymphoma (NPM–ALK)6,7,8 and more recently in adenocarcinoma of lung (EML4–ALK)9,10, is also a frequent target of genetic alteration in advanced neuroblastoma. According to our genome-wide scans of genetic lesions in 215 primary neuroblastoma samples using high-density single-nucleotide polymorphism genotyping microarrays11,12,13,14, the ALK locus, centromeric to the MYCN locus, was identified as a recurrent target of copy number gain and gene amplification. Furthermore, DNA sequencing of ALK revealed eight novel missense mutations in 13 out of 215 (6.1%) fresh tumours and 8 out of 24 (33%) neuroblastoma-derived cell lines. All but one mutation in the primary samples (12 out of 13) were found in stages 3–4 of the disease and were harboured in the kinase domain. The mutated kinases were autophosphorylated and displayed increased kinase activity compared with the wild-type kinase. They were able to transform NIH3T3 fibroblasts as shown by their colony formation ability in soft agar and their capacity to form tumours in nude mice. Furthermore, we demonstrate that downregulation of ALK through RNA interference suppresses proliferation of neuroblastoma cells harbouring mutated ALK. We anticipate that our findings will provide new insights into the pathogenesis of advanced neuroblastoma and that ALK-specific kinase inhibitors might improve its clinical outcome.

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Figure 1: Common 2p gains/amplifications and ALK mutations in neuroblastoma samples.
Figure 2: Kinase activity of ALK mutants and their downstream signalling.
Figure 3: Oncogenic role of ALK mutations.

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GenBank/EMBL/DDBJ

Data deposits

The nucleotide sequences of ALK mutations detected in this study have been deposited in GenBank under the accession numbers EU788003 (K1062M), EU788004 (T1087I), EU788005 (F1174L; TTC/TTA), EU788006 (F1174L; TTC/TTG), EU788007 (F1174C), EU788008 (F1174V), EU788009 (F1245L) and EU788010 (R1275Q). The copy number data as well as the raw microarray data will be accessible from http://www.ncbi.nlm.nih.gov/geo/ with the accession number GSE12494.

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Acknowledgements

We thank H. P. Koeffler for critically reading and editing the manuscript. We also thank M. Matsumura, Y. Ogino, S. Ichimura, S. Sohma, E. Matsui, Y. Yin, N. Hoshino and Y. Nakamura for their technical assistance. This work was supported by the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency and by a Grant-in-Aid from the Ministry of Health, Labor and Welfare of Japan for the third-term Comprehensive 10-year Strategy for Cancer Control.

Author Contributions Y.C., Y.L.C. and J.T. contributed equally to this work. M.K. and M.Sa. performed microarray experiments and subsequent data analyses. Y.C. and J.T. performed mutation analysis of ALK. Y.C., Y.L.C., J.T., M.So., L.W. and H.M. conducted functional assays of mutant ALK. A.N., M.O., T.I., A.K. and Y.H. prepared tumour specimens and were involved in statistical analysis. A.N., Y.H., H.M., J.T. and S.O. designed the overall study, and S.O. and J.T. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Yasuhide Hayashi or Seishi Ogawa.

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Chen, Y., Takita, J., Choi, Y. et al. Oncogenic mutations of ALK kinase in neuroblastoma. Nature 455, 971–974 (2008). https://doi.org/10.1038/nature07399

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