Cell culture andDrosophilamodel systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma

Damini Chand,Miki Ohira,Yasuo Yamazaki,Bengt Hallberg,Kristina Ruuth,Olena Morozova,Ruth H Palmer,Christina Schönherr,Marco A Marra,John Maris,Akira Nakagawara,Per-Erik Sandström,Edward F Attiyeh,Tommy Martinsson,Per Kogner

doi:10.1242/dmm.010348

Abstract

SUMMARYNeuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALKF1174 mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALKF1174l, (ii) kinase-dead ALK mutants, e.g. ALKI1250T (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.

Highlights

Neuroblastoma is a common childhood cancer that arises in the tissues of the sympathetic nervous system (Maris et al, 2007)
Their studies aimed to determine whether disease-associated anaplastic lymphoma kinase (ALK) mutations are universally gain-of-function and whether certain ALK mutations can confer resistance to tyrosine kinase inhibitors
Their results, together with existing data, suggest that the neuroblastoma-associated ALK mutations characterised far fall into three classes: (i) gain-of-function, ligand-independent mutations of varying activation strength, such as ALKF1174, (ii) kinase-dead ALK mutants, such as ALKI1250T and (iii) ALK mutations that are ligand-dependent and might represent ‘passenger’ mutations

Summary

Introduction

Neuroblastoma is a common childhood cancer that arises in the tissues of the sympathetic nervous system (Maris et al, 2007). It most commonly originates in the adrenal glands, but can develop at additional sites in the neck, chest and abdomen. It is considered to be a disease of developing tissue because it originates from precursor cells of neural crest tissue that are active during embryonic development. This in part explains the median age of 17 months for occurrence of neuroblastoma (Maris, 2010). We have addressed the important issue of whether ALK mutations are resistant to the medicine that is currently in use

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