Regulation and inhibition of the anaplastic lymphoma kinase
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions in regulatory regions of the intracellular catalytic domain of the intact ALK receptor tyrosine kinase (F1174L and R1275Q). The identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. We assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines expressing mutated (F1174L or R1275Q) or wild-type (overexpressed) ALK. Growth of cells expressing R1275Q-mutated ALK or high levels of wild-type ALK (due to genomic amplification) was inhibited by crizotinib, whereas cells expressing F1174L-mutated ALK were resistant. These in vitro observations were recapitulated in xenograft studies of crizotinib sensitivity. Through detailed biochemical analyses, we show that the reduced susceptibility to crizotinib inhibition of F1174L-mutated ALK results from an increased ATP-binding affinity (by Km, ATP measurement), as also seen in the development of resistance to EGFR inhibitors in non-small cell lung cancer by 'gatekeeper' residue mutation. In addition to mutations observed at the R1275 and F1174 positions, our sequencing efforts have defined the full spectrum of ALK mutations in a panel of 1597 primary neuroblastoma tumor samples. We extended our biochemical analysis to these mutations. Our results indicate that many of these novel mutation variants are constitutively activating, developing our structural understanding of the ALK catalytic domain and suggesting that they are clinically relevant. We conclude that, in several cases, the in vitro crizotinib sensitivity of these variants correlate with Km, ATP values, like R1275Q and F1174L. However, inhibitor affinity also appears to be altered for select mutant variants. These results have direct relevance to the future development and clinical application of inhibitors for ALK-driven malignancies, and suggest a need for higher-affinity ALK inhibitors.