Neuroblastoma-associated ALK variants have distinct cellular and biochemical activities

Abstract

Mutations in anaplastic lymphoma kinase (ALK) are associated with high-risk neuroblastoma, a childhood cancer arising in trunk neural crest cells. The role of ALK in undifferentiated NC is still unknown; however, the presence of activating mutations in ALK correlates with migratory and invasive cell behaviours in neuroblastoma cell lines. Here, we show the functional consequences of ALK overexpression on neural crest cells, by comparing wildtype ALK (ALKWT) protein to ALK gain-of-function variants ALKF1174L and ALKR1275Q. Elevated ALK activity, independent of mutational status leads to increased migration velocity and loss of directionality, while ALKF1174L overexpression presents additional effects on cytoskeletal protrusions. These results correlate with increased binding of ALKF1174L to GSK3, which has previously been shown to regulate cytoskeletal dynamics in neural crest cells. Further, molecular dynamics simulations of the ALK-GSK3 complex show high flexibility, suggestive of enhanced allosteric regulation. Together, our data show that activating mutations in ALK drive migratory changes in trunk NC cells, potentially mediated by its novel interacting partner GSK3. Significance StatementAnaplastic lymphoma kinase (ALK)-associated neuroblastoma phenotypes arise in the neural crest lineage, but the effects on neural crest cell behaviours are not well-studied. Here, we use primary mouse neural crest cells to study the functional relevance of ALK-activating mutations on cell migration, which we then biochemically link to interactions with GSK3-isoforms. We also directly compare the activities of wild-type ALK with two gain-of-function variants associated with metastatic disease, ALKF1174L and ALKR1275Q, and find that ALKF1174L causes increased cytoskeletal protrusions and GSK3 binding. Our study provides insights into a signalling pathway in neural crest that drives cytoskeletal rearrangements and pathological migration in neuroblastoma.