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

Abstract

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.

Oncogenic protein condensates suppress growth factor perception and modulate drug tolerance

“…We then asked how Grb2 localization might change in the presence of ALKi ( Figure 4E ). ALKi caused Grb2 puncta to rapidly dissolve into the cytoplasm (t 1/2 = 17 +/- 2 min) ( Figure 4F,G, Supplementary Movie 2 ), in line with recent reports (Sampson et al, 2021). Finally, sequential treatment with ALKi and EGF allowed robust membrane translocation of Grb2 even in the presence of EML4-ALK ( Figure 4H,I ).…”

A Polytherapy Strategy Using Vincristine and ALK Inhibitors to Sensitise EML4-ALK-Positive NSCLC

“…In our data, the short EML4-ALK V3 fusion had higher microtubule nucleation capacity compared to the long EML4-ALK V1 protein. This is not surprising, given our recent discovery of a specific region within the partial TAPE domain within the EML4-ALK V1 fusion protein that suppresses microtubule binding [10].…”

“…Since EML4-ALK V3, but not V1, is sequestered to microtubules in the presence of ALK-TKIs [10], we tested whether the addition of vincristine could enhance the cytotoxicity of ALK-TKI (crizotinib or ceritinib) in ALK-positive lung cancer cell lines. The combination of crizotinib plus vincristine (1 nM) was more effective in reducing cell viability than crizotinib alone with ~5-fold lower IC 50 in H3122 cells (Figure 2A).…”

“…All EML4-ALK fusion variants have the N-terminal trimerization domain (TD) and some also contain the basic region of EML4, which together are essential for microtubule binding [9]. Recently, it has been reported that the ALK inhibitors, ceritinib and lorlatinib, sequester inactive EML4-ALK V3 protein to microtubules, whereas V1 remains in the cytoplasm [10]. The C-terminal TAPE domain of EML4 consists of a set of 14 individual sub-domains that form a tandem pair of 7-bladed beta-propellers [11].…”

“…The C-terminal TAPE domain of EML4 consists of a set of 14 individual sub-domains that form a tandem pair of 7-bladed beta-propellers [11]. The presence of the 12N blade subdomain in EML4-ALK V1 protein, which is missing from V3, prevents the catalytically inactive protein from binding to microtubules [6,10].…”