The mesenchymal morphology of cells expressing the EML4-ALK V3 oncogene is dependent on phosphorylation of Eg5 by NEK7

EML4-ALK oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancer patients. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as V1. Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis and poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3:NEK9:NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain to be identified. Here, we show that the microtubule-based kinesin motor protein, Eg5, is recruited to interphase microtubules in response to expression of EML4-ALK V3, while chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of NEK7 interferes with recruitment of Eg5 to microtubules in cells expressing EML4-ALK V3 and results in reduced cell length, but this is reversed by co-expression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Taken together, we propose Eg5 acts as a substrate of NEK7 in cells expressing EML4-ALK V3 and that Eg5 phosphorylation promotes the altered morphology typical of these cells.