Fig. 4: EML4-ALK assemblies suppress EGFR through sequestration of RTK effectors.

A Quantification of ppERK response to EGF (50 ng/mL) in Beas2B cells that were transfected with EML4-ALK (V1), EML4-ALK (∆TD), or kinase-dead EML4-ALK (K589M), or with an empty vector (EV). Data points represent mean ± SEM of three biological replicates, each representing 200–400 EV cells, 130-270-EML4-ALK (V1) cells, 55–160 EML4-ALK (∆TD) cells, or 260–450 EML4-ALK (K589M) cells. B Colocalization of EML4-ALK condensates with endogenously tagged GRB2 (GRB2:mNG2). See Fig. 5 for quantitation. Scale = 10 µm. C GRB2:mNG2 Beas2B cells were transiently transfected with EML4-ALK and stimulated with EGF (50 ng/mL) to visualize GRB2 translocation in the presence and absence of EML4-ALK (V1). D Impaired membrane translocation of GRB2 in the presence of EML4-ALK condensates. Time in mm:sec. See Supplementary Movie 1. E Line scan of GRB2 intensity distribution in the presence (red) or absence (gray) of EML4-ALK expression, as depicted in (C). F Quantitation of translocation of endogenous GRB2 or SOS1 in the presence (red) or absence (gray) of EML4-ALK. Boxplot indicates the median and upper/lower quartiles, and whiskers extend to 1.5*IQR. See Fig. 5 for full quantitation. G GRB2 localization and translocation were visualized upon treatment with 1 µM ALKi and subsequent stimulation with EGF (50 ng/mL). Time in hh:mm. H Quantification of kinetics of GRB2 dissociation from condensates after ALKi treatment. I ALKi restores GRB2 and SOS translocation. Plot shows median translocation of endogenous adapters in cells expressing EML4-ALK(V1) represented as a fraction of translocation in the absence of EML4-ALK (V1). Data represent medians, error bars show 1st and 3rd quartiles of 1000 bootstrapped samples (distributions found in Fig. 5F, G). Significance assessed by one-sided bootstrap test for comparison of medians. See Fig. 5F, G for underlying data and quantitation. J Immunoprecipitation of EGFR shows enhanced co-precipitation of GRB2 and SOS1 in the presence of both ALKi pretreatment and EGF in STE-1 cells. gray arrows: non-specific bands. K Densitometry quantification of three independent pulldowns. L Testing effect of GRB2 overexpression on ERK response. M Expression levels of GRB2-GFP or GFP analyzed in (N, O). N ppERK levels in the absence (open circles) or presence (closed circles) of EGF stimulation (50 ng/mL) as a function of expression levels of GFP or GRB2-GFP. Data represent mean ± SEM of three biological replicates, each representing the mean of 100–300 cells. O Absolute magnitude of ppERK increase for each expression bin from data shown in (N). Significance assessed by one-sided T-test, n = 3 biological replicates. P Fold-change of response calculated from data in (N). Q Conceptual model of how EML4-ALK suppresses transmembrane RTKs. EML4-ALK sequesters adapters like GRB2/SOS1 and prohibits their translocation to activated RTKs. ALK inhibition releases adapter sequestration and restores cellular response to RTK stimulation.