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

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.

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK positive neuroblastoma cells, Drosophila and mice

“…However, brigatinib inhibits ALK addicted neuroblastoma cell line proliferation with roughly 2 to 3-fold lower IC 50 values compared with crizotinib. Previously described expression of gain-of-function "hot spot" ALK mutations ALK-F1174L and ALK-R1275Q in Drosophila eye imaginal discs with the Gal4-UAS-system results in a rough eye phenotype that is 100% penetrant [7,38,40]. Offspring that were grown on food containing brigatinib displayed a concentration dependent improvement of the rough eye phenotype.…”

“…The expression of human ALK proteins was confirmed by immunostaining of eye discs using antihuman ALK antibody ( Supplementary Figure 2A, 2B). Ectopic expression of the ALK gain-of-function alleles F1174L and R1275Q led to disrupted eye morphology in all offspring (100%), a so called 'rough eye' phenotype, which is characterized by disorganized ommatidia and missing interommatidial bristles, reflecting their robust ligand-independent activity ( Figure 3) [7,38,40]. All larvae that were grown on brigatinib inhibitor-containing food developed into flies which showed a concentrationdependent improvement of the rough eye phenotype (Figure 3).…”

“…6 ) co-transfected, in 100 μl of Ingenio electroporation solution (Minus Bio LCC), with pEGFPN1 (Clonetech) (0.5 ng) and ALK-mutant (0.75 ng) or ALK-wt (0.75 ng) as indicated were seeded sparingly into 24-well plates [38]. ALK-wild type was stimulated with 1 μg/ml mAb31.…”

Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice

“…The F1174 residue lies in the alpha C helix [3] and it has been suggested that a F to L change will result in an increased affinity for ATP [20]. The F1174L mutation has been shown to be a ligand-independent activating mutation [21]. The L and V residues present with the same physicochemical properties and are expected to induce the same consequences on the receptor's activity.…”

ALK-Positive Cancer: Still a Growing Entity

“…It was also proven that ALK antagonists, such as crizotinib, may effectively block neurite outgrowth in a ligand-independent manner. They also decreased the activity of all types of mutant ALK receptors, thus inhibiting the ALKdependent signal transduction pathways [119]. The response to the inhibitors depends on the dose and the type of ALK mutation, with F1774L being the most resistant to the drug and with complete regression of the xenograft in cases with ALK amplification or R1275Q mutation [120].…”