Different EML4-ALK fusion variants and novel ALK resistance mutations induce differential sensitivity to ALK kinase inhibitors

EML4-ALK positive lung cancer can today effectively be treated with the ALK kinase inhibitor crizotinib. However, not only the initial tumor response to crizotinib treatment is heterogeneous, but also several resistance mutations that limit treatment efficiency have been described. In this study, cells expressing different EML4-ALK variants were shown to exhibit differential sensitivity to structurally unrelated ALK kinase inhibitors. Interestingly, these variants also exhibited differential HSP90 inhibitor sensitivity, however, with a varying distribution across the fusion variants. Furthermore, combining ALK and HSP90 inhibitors induced synergistic cytotoxicity in all ALK fusion gene expressing cells, arguing for a functional link between HSP90 and the ALK fusion protein. Most likely, this functional interaction is based on the ALK fusion partner induced protein instability, which can be enhanced by kinase inhibitors, and attenuated by HSP90 binding. As opposed to these ALK inhibitor scaffold independent differences in sensitivity, cells expressing the crizotinib resistance mutations L1196M, F1174L or G1269S were highly sensitive to TAE684 treatment. Furthermore, two orthogonal mutagenesis screens identified two novel resistance mutations (L1198P and D1203N), which induced a high level of resistance to crizotinib and TAE684. The L1198P mutation most likely induced resistance to crizotinib by shifting the kinase equilibrium to a more active conformation and consequently hampering the inhibitor binding to the kinase domain. Resistance to TAE684 was most likely induced by mutation induced structural alterations at the hinge region of the kinase, impeding the interactions with the methoxy-group of TAE684. The exact mechanism of resistance induced by D1203N is unclear. The described mechanisms of differential sensitivity might explain some of the heterogeneous responses of EML4-ALK positive tumors after treatment with crizotinib. Furthermore, these findings demonstrate that the ALK genotype as well as the choice of ALK inhibitor highly impacts the therapeutic efficiency and should be taken into account for targeted therapy in ALK positive lung cancer and other cancers with ALK aberrations.


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