Cell culture and Drosophila model systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma

Abstract

Neuroblastoma 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 ALK(F1174) mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALK(F1174l), (ii) kinase-dead ALK mutants, e.g. ALK(I1250T) (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.

Fig. 1.

Domain structure of ALK mutations and their activation of downstream targets. (A) The extracellular, transmembrane domain and the intracellular domain, which contains the protein tyrosine kinase domain are shown. Mutation residues within the intracellular domain are indicated by numbers. Also indicated are the A-loop (blue), ATP-binding loop (P-loop, green) and catalytic loop (yellow). The scheme is not drawn to scale. (B) ALK kinase domain showing locations of found mutations (red balls) from neuroblastoma patients. Also indicated are the A-loop (blue), ATP-binding loop (green) and catalytic loop (yellow). The figure was generated with PyMol using published coordinates (PDB#3LCS) (Bossi et al., 2010). (C) PC12 cells expressing human wild-type and mutant ALKs were serum-starved for 48 hours prior to stimulation with 1 μg/ml of mAb31 for 30 minutes. Pre-cleared cell lysates were analysed on SDS/PAGE followed by western blotting using the indicated antibodies. ALK, pSTAT3, pERK and pan-ERK antibodies were employed as loading controls.

Fig. 2.

Crizotinib abrogates ALK-mediated neurite outgrowth in PC12 cells. PC12 cells were co-transfected with pcDNA3-human ALK and pEGFPN1 and seeded out in the presence or absence of crizotinib. Cells were stimulated with 1 μg/ml mAb31. After 48 hours, cells were scored for neurite outgrowth. The experiment was carried out in triplicate and each sample within an experiment was performed in duplicate (error bars indicate s.d.). Black bars represent untreated cells, grey bars denote cells stimulated with mAb31 and white bars indicate cells treated with 250 nM crizotinib on its own or together with mAb31.

Fig. 3.

Expression of human ALK in Ba/F3 cells. (A) Ba/F3 cells were transfected with human wild-type or mutant ALKs and selected with G418 in the presence of IL-3. Whole cell lysates were prepared and run on SDS-PAGE, followed by immunoblotting with indicated antibodies. (B) After selection in the presence of IL-3 and G418, Ba/F3 cells were washed and seeded at 0.5×10⁶ cells/ml into IL-3-free media containing G418. Cells were counted at different time points using the Trypan Blue exclusion method. (C) Ba/F3 cells expressing ALK were treated with the indicated concentrations of crizotinib in the presence of IL-3. Proliferation was assayed using resazurin. The results obtained indicate that crizotinib does not cause any toxicity to cells at the concentrations used. Points show the increase in relative fluorescence from cells with IL-3 compared with the relative fluorescence from cells without IL-3 and crizotinib (set at a value of 100). Each sample was analysed in triplicate.

Fig. 4.

Crizotinib inhibits both human ALK^F1174-driven proliferation and phosphorylation at Tyr1278 and Tyr1604 of ALK in Ba/F3 cells. (A) IC₅₀ values for crizotinib for both human ALK^F1174I and human ALK^F1174L were calculated. Values represent mean + s.d. from at least three independent experiments. (B) IL-3-independent Ba/F3 cells expressing human ALK^F1174Iand human ALK^F1174L were treated with different concentrations of crizotinib. Cell viability was assayed using resazurin after 3 days. (C) Ba/F3 whole cell lysates were separated with SDS/PAGE followed by immunoblotting with the indicated antibodies. ALK activation, for 3 hours, was checked using phosphospecific antibodies against Tyr1278 (p-ALK Y1278) and Tyr1604 (p-ALK Y1604). ERK phosphorylation was checked using phospho-specific antibody (p-ERK). Loading control was pan-ERK.

Fig. 5.

Transformation potential of human ALK mutants. (A,B) The transforming potential of the mutations was assayed by transfecting NIH3T3 cells with pcDNA3-human ALK (mutant and wild type). Cells were selected with G418 for 3 weeks. (A) Representative images of foci. Cells were stained with crystal violet and the number of foci were counted. (B) Bars represent average number of foci per transfection ± s.d. from three independent transfections performed in triplicates.

Fig. 6.

Ectopic expression of human ALK^A1234T and human ALK^R1464STOP in Drosophila eye does not generate the rough eye phenotype. Adult fly eyes (upper) and larval eye discs (lower) ectopically expressing mutant human ALK variants. Human ALK^wt and human ALK^F1174L, ALK^F1174L, ALK^A1234T and ALK^R1464STOP were expressed in the Drosophila eye with pGMR-Gal4. The gain-of-function mutation ALK^F1174L generated a rough eye phenotype, in contrast to both ALK^A1234T and ALK^R1464STOP. Eye discs were stained with anti-human ALK antibody (red) to confirm protein expression.