Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation

Variants of the oncogenic EML4-ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4-ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4-ALK V3 but not V1 protein re-localises to microtubules, an effect recapitulated in a catalytically inactive EML4-ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild-type and catalytically inactive EML4-ALK V3 proteins, but not a Lys-Glu salt bridge mutant. We propose that EML4-ALK foci formation occurs as a result of transient association of stable EML4-ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4-ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors. Synopsis EML4-ALK fusion proteins phase separate into foci that contain oncogenic signaling factors, through a mechanism that depends on an active ALK kinase conformation. Variants have distinct dynamics determined by a specific region of EML4. EML4-ALK V1 and V3 form cytoplasmic foci that contain signaling proteins of the MAPK, PLCγ and PI3K pathways. Active kinase domain and EML4 TD interactions underpin dynamic V3 foci, further interactions make V1 foci static. ALK inhibitors ceritinib and lorlatinib dissolve foci and EML4-ALK V3 but not V1 re-localises to microtubules. Dissolving EML4-ALK V3 foci inhibits ALK signalling, which may provide a new strategy for targeting aggressive NSCLC. Graphical Abstract EML4-ALK fusion proteins phase separate into foci that contain oncogenic signaling factors, through a mechanism that depends on an active ALK kinase conformation. Variants have distinct dynamics determined by a specific region of EML4.