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

2021; Springer Nature; Volume: 22; Issue: 12 Linguagem: Inglês

10.15252/embr.202153693

1469-3178

Josephina Sampson, Mark W. Richards, Jene Choi, Andrew M. Fry, Richard Bayliss,

Microtubule and mitosis dynamics

Article18 October 2021Open Access Source DataTransparent process Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation Josephina Sampson Josephina Sampson orcid.org/0000-0002-0147-6014 School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK Search for more papers by this author Mark W Richards Mark W Richards orcid.org/0000-0003-1108-2825 School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK Search for more papers by this author Jene Choi Jene Choi Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Search for more papers by this author Andrew M Fry Andrew M Fry orcid.org/0000-0003-4417-7329 Department of Molecular and Cell Biology, University of Leicester, Leicester, UK Search for more papers by this author Richard Bayliss Corresponding Author Richard Bayliss [email protected] orcid.org/0000-0003-0604-2773 School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK Search for more papers by this author Josephina Sampson Josephina Sampson orcid.org/0000-0002-0147-6014 School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK Search for more papers by this author Mark W Richards Mark W Richards orcid.org/0000-0003-1108-2825 School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK Search for more papers by this author Jene Choi Jene Choi Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Search for more papers by this author Andrew M Fry Andrew M Fry orcid.org/0000-0003-4417-7329 Department of Molecular and Cell Biology, University of Leicester, Leicester, UK Search for more papers by this author Richard Bayliss Corresponding Author Richard Bayliss [email protected] orcid.org/0000-0003-0604-2773 School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK Search for more papers by this author Author Information Josephina Sampson1, Mark W Richards1, Jene Choi2, Andrew M Fry3 and Richard Bayliss *,1 1School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK 2Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea 3Department of Molecular and Cell Biology, University of Leicester, Leicester, UK *Corresponding author. Tel: +0113-34-39919; E-mail: [email protected] EMBO Reports (2021)22:e53693https://doi.org/10.15252/embr.202153693 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. 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. Introduction Lung cancer is the most common cancer globally and, despite advances in targeted treatment, the 10-year survival rate of patients with lung cancer is only 9% in the UK (Sabir et al, 2017). More than 80% of lung cancers are classified as non-small-cell lung cancer (NSCLC), of which most are adenocarcinomas. While activating mutations in RAS or epidermal growth factor receptor (EGFR) are the most common drivers of NSCLC, oncogenic fusions created by chromosome translocations are also frequent driver events (Yuan et al, 2019). These frequently encode proteins in which the kinase domain of a receptor tyrosine kinase (RTK) is constitutively activated by translational fusion to a self-associating region of another protein. One common example is the fusion of anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4), found in ˜ 2–9% of NSCLC patients (Soda et al, 2007). To date, at least 15 EML4-ALK variants have been identified all containing the cytoplasmic tyrosine kinase domain of ALK. The variants differ in the point of fusion within the EML4 gene although all retain the N-terminal region of EML4 that encodes its coiled-coil trimerisation domain (TD) through which the fusion proteins achieve self-association and constitutive ALK activation (Soda et al, 2007; Richards et al, 2014). The predominant EML4-ALK variants identified in patients are variants 1 and 3 (V1 and V3), accounting for around 33% and 29% of cases, respectively. Structurally, EML4-ALK V3 contains the TD and an unstructured, basic region that mediates microtubule association of EML4, but lacks its entire C-terminal tandem atypical β-propeller (TAPE) domain, whereas V1 also retains a large, albeit incomplete, part of the TAPE domain, the presence of which confers dependence on Hsp90 for stability (Richards et al, 2014). Since EML4-ALK fusions were identified in NSCLC, potent tyrosine kinase inhibitors (TKI) against ALK have been developed and used as a first-line treatment for those patients. First- and second-generation inhibitors, crizotinib, ceritinib and alectinib, are currently used as first-line treatment for ALK-positive NSCLC patients, while lorlatinib, a third-generation inhibitor, is approved for a second- or third-line treatment after crizotinib, ceritinib or alectinib in ALK-positive metastatic NSCLC (Solomon et al, 2014, 2018). NSCLC harbouring different EML4-ALK variants exhibit different responses to ALK inhibitors. NSCLC patients identified with EML4-ALK V1 respond to crizotinib and have increased progression-free survival (PFS) after treatment (Shaw et al, 2011; Yoshida et al, 2016). However, EML4-ALK V3-positive NSCLC patients demonstrate a higher metastatic spread and increased aggressiveness of the disease, while in vitro NSCLC cells harbouring EML4-ALK V3 exhibit resistance to various ALK inhibitors (Woo et al, 2017; Christopoulos et al, 2018; O'Regan et al, 2020). The cytoplasm is no longer considered a homogenous solution but rather to contain unevenly distributed protein and RNA molecules forming dynamic assemblies through transient molecular interactions. This behaviour sometimes causes molecules associated with these dynamic assemblies to partition from the bulk cytoplasm into droplets through a process called liquid–liquid phase separation (LLPS) (Hyman et al, 2014; Boeynaems et al, 2018). Several studies have reported proteins forming foci within the cytoplasm, such as Tau and polo-like kinase 4 (PLK4) (Wegmann et al, 2018; Park et al, 2019). Indeed, liquid-like foci have long been described in numerous papers as membraneless organelles, such as the P granules in C. elegans embryos (Brangwynne et al, 2009). The major characteristics of LLPS foci are that they: (i) fuse after touching and revert into a spherical shape; (ii) deform, diffuse and exchange material with the cytoplasm; (iii) adopt a spherical shape that is driven by surface tension; and (iv) recover rapidly through internal rearrangement and cytoplasmic exchange when photobleached (Hyman et al, 2014). Recently, Tulpule and colleagues have observed RTK fusion proteins, including EML4-ALK V1 and V3, forming membraneless cytoplasmic granules that act as centres for the organisation and activation of RAS and other downstream signalling pathway components (Tulpule et al, 2021). Intriguingly, the granules are disrupted by a catalytically inactive mutant leading to the hypothesis that their formation is dependent on ALK activity (Tulpule et al, 2021). Despite the recent progress made to understand the functional role of EML4-ALK foci in oncogenic signalling, the molecular interactions that underpin their formation are unknown. In the present study, we demonstrate that EML4-ALK V1 and V3 proteins partition into cytoplasmic foci in a manner dependent on the active conformation, rather than catalytic activity itself, of the ALK kinase domain. We confirm that the V1 and V3 cytoplasmic foci are rich in protein–protein interactions and capture signalling proteins such as GRB2, SOS1, PLCγ2, PI3K and KIT, suggesting that they may act as hubs for activation of downstream pathways. Interestingly, while V3 foci exhibit typical LLPS characteristics, we found that V1 foci have a more solid-like state due to the presence of a specific sub-domain within the partial TAPE region of V1. We also reveal how the ALK inhibitors, ceritinib, alectinib and lorlatinib, differentially affect the localisation and behaviour of EML4-ALK V1 and V3 inside the cell by dissolving or maintaining these cytoplasmic foci. Taken together, we propose a model to explain how EML4-ALK foci formation is connected to the activation mechanism of ALK. Results EML4-ALK V3 forms dynamic cytoplasmic foci while V1 foci are static Previous studies have reported distinct subcellular localisation of EML4-ALK to cytoplasm or microtubules depending on the type of variant; however, the precise localisation of these proteins was not clearly defined (Hrustanovic et al, 2015; Richards et al, 2015). We therefore examined the subcellular localisation of EML4-ALK V1 and V3 by fixed-cell imaging in several cell lines: NSCLC patient-derived cancer cell lines harbouring endogenous EML4-ALK V1 (H3122) or EML4-ALK V3 (H2228), a non-transformed human lung epithelial cell line (Beas2B) expressing EML4-ALK V1 or V3 in a doxycycline-inducible manner and HEK293 cells overexpressing YFP-EML4-ALK V1 or V3. Fixed imaging analysis revealed approximately 15-60 prominent cytoplasmic foci of EML4-ALK V1 in each of the three different cell types (Figs 1A and B, and EV1A and B). Similarly, we identified 25–50 prominent foci of EML4-ALK V3 in the cytoplasm of each different cell type (Figs 1C and D, and EV1A and B). In contrast, YFP-ALK 1058–1620 (the region of ALK present in the EML4-ALK fusions) showed very few (at most 5) distinct droplets in the cytoplasm upon expression in HEK293 cells (Fig EV1C and D). To test whether formation of EML4-ALK V1 and V3 cytoplasmic foci requires catalytic activity of the ALK tyrosine kinase, we generated catalytically inactive (D1270N) versions of V1 and V3 and analysed their localisation by fixed imaging. Observation of transfected HEK293 cells revealed significantly fewer distinct EML4-ALK V1 and V3 cytoplasmic foci with the kinase-dead (KD) compared with wild-type (WT) constructs (Fig 1A–D). Interestingly, the KD mutant of V3, but not V1, associated strongly with what appeared to be bundled microtubules (Fig 1A–D). Taken together, these data suggest that both EML4-ALK variants form foci of concentrated protein in the cytoplasm in a process that requires ALK catalytic activity. Figure 1. EML4-ALK V1 and V3 activation induces the formation of cytoplasmic foci H3122 cells expressing endogenous EML4-ALK V1 and HEK293 transfected with YFP-EML4-ALK V1 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. Violin plots showing the number of EML4-ALK V1 cytoplasmic foci per cell. Data represent 30–50 counts from three biological replicates. ***P < 0.001, ****P < 0.0001 in comparison with HEK293 YFP-EML4-ALK-V1 KD by one-way ANOVA. H2228 cells expressing endogenous EML4-ALK V3 and HEK293 transfected with YFP-EML4-ALK V3 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red) and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. Violin plots showing the number of EML4-ALK V3 cytoplasmic foci per cell. Data represent 30–50 counts from four biological replicates. ***P < 0.001, ****P < 0.0001 in comparison with HEK293 YFP-EML4-ALK-V3 KD by one-way ANOVA. Time-lapse imaging of transfected HEK293 YFP-EML4-ALK V1 WT to observe the movement of cytoplasmic foci. Representative still images are shown of an area of cytoplasm containing YFP-EML4-ALK V1 WT and V3 WT foci at the times indicated. Scale bar, 1 μm. Time-lapse imaging of transfected HEK293 YFP-EML4-ALK V3 WT to observe the movement of cytoplasmic foci. Representative still images are shown of an area of cytoplasm containing YFP-EML4-ALK V1 WT and V3 WT foci at the times indicated. Scale bar, 1 μm. Kymographs showing the movement of YFP-EML4-ALK V1 WT, YFP-EML4-ALK V3 WT and YFP-EML4-ALK V3 KD over the duration of the observation period. Scale bars; 1 μm horizontal; seconds vertical. Whisker plot showing the calculated velocity of single events in the kymograph. Data represent 20 counts from 10 kymographs. n = 2. Error bar represents SD of two biological replicates. *P < 0.05 in comparison with YFP EML4-ALK V3 KD by unpaired t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Beas2B inducible cells form cytoplasmic foci but not YFP-ALK, and the tracking analysis of EML4-ALK V3 foci Inducible Beas2B V1, V3 and PVX (empty vector) cells were stained with either anti-ALK (green) or anti-GFP (green), anti-α-tubulin (red) and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. Violin plot showing the number of cytoplasmic foci per cell. Data represent 40–50 counts from three biological replicates. ****P < 0.0001 in comparison with Beas2B PVX (empty vector) by one-way ANOVA. HEK293 cells were transfected with YFP-ALK 1,058–1,620 for 48 h before fixation and staining with anti-GFP (green), anti-α-tubulin (red) and DAPI (blue). Scale bars, 10 μm. Violin plot representing the number of cytoplasmic foci per cell. Data represent counts from at least 20 cells from two biological replicates. Not significant (ns) in comparison with YFP EML4-ALK 1,058–1,620 by unpaired t-test. Track classification of YFP-EML4-ALK V3 foci in the cytoplasm of HEK293. Duration of the movie was 12 s. Scale bar, 1 μm. Each coloured and numbered trajectory indicates the movement of an individual droplet. Heatmap representing the number of split and merge events in each trajectory. Plot of foci maximum speed (μm/s) versus foci diameter (μm). The best-fit line shows correlation with 95% confidence interval. Plot displays the total distance (μm) and displacement (μm) of each trajectory. Colours and numbers relate to the tracks shown in (E). Download figure Download PowerPoint To explore the properties of the EML4-ALK V1 and V3 cytoplasmic foci, we performed time-lapse imaging of transfected YFP-EML4-ALK V1 and V3 HEK293 cells. Observation of YFP-EML4-ALK V1 WT transfected cells revealed a lack of movement of V1 cytoplasmic foci, in contrast to rapid movements of individual YFP-EML4-ALK V3 WT foci (Fig 1E and F). While live-cell imaging confirmed the presence of distinct cytoplasmic foci in HEK293 cells transfected with YFP-EML4-ALK V1 and V3 WT (Movie EV1 and EV3), these were not detected with the KD construct of each variant (Movie EV2 and EV4). As observed in fixed cells, EML4-ALK V1 KD cells showed a dispersed localisation whereas the V3 KD protein gave a robust microtubule association (Movie EV2 and EV4). Further analysis suggested that YFP-EML4-ALK V3 WT foci had the ability to split and coalesce frequently and to diffuse in a random manner in the cytoplasm with speeds in the range of 0.5–3 μm/s that inversely correlated with foci size (Fig EV1E–H). Furthermore, kymographs revealed that WT V3 foci moved erratically at a mean velocity of 0.7 μm/s, while WT V1 cytoplasmic foci moved smoothly at a mean velocity of 0.3 μm/s (Fig 1G and H). Similar to WT V1, kinase-dead (KD) V3 drifted smoothly at a mean velocity of 0.2 μm/s (Fig 1G and H). Taken together, these data indicate that EML4-ALK V3 cytoplasmic foci are motile and able to merge and split, in contrast to static V1 cytoplasmic foci that behave as solid granules. Cytoplasmic EML4-ALK V1 and V3 foci act as signalling hubs We next investigated whether the cytoplasmic EML4-ALK V1 and V3 foci are enriched in cytoplasmic components that are required for oncogenic, downstream signalling pathways. Since receptor tyrosine kinase (RTK) signalling is controlled by lipid-ordered plasma membrane microdomains (Delos Santos et al, 2015), we examined whether EML4-ALK V1 and V3 cytoplasmic foci are associated with lipid membranes by carrying out subcellular fractionation of H3122 and H2228 in the presence of detergent. Unlike the control protein, calnexin, which is an internal membrane protein, neither EML4-ALK V1 nor V3 were solubilised in the presence of 1% Triton X-100 treatment, but rather behaved similarly to DCP1B, a cytoplasmic ribonucleoprotein granule from the P-body (Appendix Fig S1A–D). These data suggest that EML4-ALK V1 and V3 foci are not membrane enclosed structures. In addition, we investigated whether EML4-ALK V1 and V3 cytoplasmic foci are formed by multivalent interactions of protein–RNA complexes by carrying out subcellular fractionation of H3122 and H2228 cell lysates with RNase A. EML4-ALK V1 and V3 fractionation was unaltered by RNase A treatment while, DCP1B, which is a ribonucleoprotein, was partially shifted to the supernatant fraction (Appendix Fig S1E–H). Since the behaviour of EML4-ALK V1 and V3 upon subcellular fractionation was independent of lipid or RNA, it is likely that the formation of cytoplasmic foci of EML4-ALK V1 and V3 is driven primarily by protein–protein interactions. As proteomic analyses and cellular assays have identified several cellular networks through which EML4-ALK can signal (Hrustanovic et al, 2015; Zhang et al, 2016), we chose to look for components of the RAS/MAPK, PI3K/AKT and stem cell factor (SCF)/KIT signalling pathways that colocalised within those EML4-ALK cytoplasmic foci. Using immunofluorescence microscopy, we observed the colocalisation of MAPK pathway adaptor proteins, GRB2 and SOS1, with EML4-ALK V1 and V3 cytoplasmic foci in the patient-derived H3122 and H2228 cells, and in the inducible Beas2B cells (Figs 2A and B, and EV2A–D). Along with the GRB2 and SOS1 proteins, EML4-ALK V1 and V3 foci were locally enriched with other kinases, notably c-KIT (phosphorylated on Tyrosine 721), the p85 beta regulatory subunit of PI3K, and PLCγ2 (phosphorylated on Tyrosine 759) in both endogenous H3122 (V1) and H2228 (V3) cells (Figs 2A and B, and EV2E–G), as well as in Beas2B V1 and V3 cells (Fig EV2A and B, E–G). Together, these data demonstrate recruitment of adaptor proteins and activated enzymes at the EML4-ALK V1 and V3 cytoplasmic foci. Figure 2. Cytoplasmic EML4-ALK V1 and V3 foci contain downstream signalling proteins A, B. H3122 and H2228 cells were stained for anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C. HEK293 cells transfected with YFP-EM4L-ALK V1 WT or V3 WT for 48 h and treated with 5% or 10% 1,6-hexanediol. Representative still images after 5 min (taken from time-lapse movies) are shown of cells treated with 10% 1,6-hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci. D. Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5–10 counts from three independent experiments (n = 3). Error bar represents SD of three biological replicates. ****P < 0.0001 in comparison with YFP-EML4-ALK V3 WT DMSO by one-way ANOVA. E. Representative Western blots of HEK293 transfected EML4-ALK V1 WT and V3 WT for 48 h. Cells were treated with either 5% or 10% 1,6-hexanediol for 5 min. DMSO was used as a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n = 2 experiments. Source Data for Figure 2 [embr202153693-sup-0004-SdataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Association of signalling protein with active EML4-ALK V1 and V3 cytoplasmic foci in inducible Beas2B cells A, B. Inducible Beas2B V1 and V3 cells were stained with anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C–G. Intensity profiles showing colocalisation between endogenous GRB2, SOS1, pC-KITY721, PI3K p85β or pPLCγ2Y759 and ALK staining in different cell lines. R (Pearson's correlation coefficient) measures the correlation between the indicated proteins and ALK signals. Pearson's measurements are from 20 foci of 10–20 cells for each antibody combination. Data in all whisker plots represent counts from at least 10 cells, n = 3. The central dashed band indicates the minimum Pearson R value (0.5) required for colocalisation. All whisker boxplots indicate the minimum and maximum Pearson R values of each cell line. *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001 in comparison of each cell line by one-way ANOVA. Download figure Download PowerPoint We then investigated the nature of the EML4-ALK V1 and V3 cytoplasmic foci using treatment with the aliphatic alcohol 1,6-hexanediol which is able to disrupt the hydrophobic interactions that drive the formation of droplets by liquid–liquid phase separation (LLPS), but which does not affect solid protein aggregation or assemblies (Kroschwald et al, 2017). Treatment with 5% or 10% 1,6-hexanediol dissolved most of the foci formed by EML4-ALK V3 but not those formed by EML4-ALK V1 (Fig 2C and D). Collectively, these data suggest that EML4-ALK V3 cytoplasmic foci behave as a form of LLPS, while EML4-ALK V1 forms static, solid foci. To determine the importance of the cytoplasmic foci on EML4-ALK downstream signalling, we tested the consequences of their dissolution by 1,6-hexanediol. Interestingly, the disruption of cytoplasmic foci by 1,6-hexanediol resulted in significant loss of activated ALK (pY1640), ERK (pT202/Y204), AKT (pS473), STAT3 (pY705), PLCγ2 (pY759) and C-KIT (pY721) in HEK293 cells expressing YFP-EML4-ALK V3 but had minimal impact in cells expressing YFP-EML4-ALK V1 (Fig 2E). The activation of downstream signalling was also significantly reduced by 1,6-hexanediol treatment in patient-derived H2228 (V3) cells, but not in H3122 (V1) cells (Fig EV3A). Click here to expand this figure. Figure EV3. Loss of signalling proteins upon 1,6-hexanediol and ALK inhibitors A. H3122 and H2228 cells were treated with either 5% or 10% 1,6-hexanediol for 5 min. DMSO was used a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data represent of n = 2 experiments. B. H2228 and Beas2B V3 cells were treated with ALK inhibitors for 4 h before fixation and staining with GRB2 and ALK antibodies for proximity ligation assay (PLA). Nuclei are indicated by DAPI staining (blue). Red foci indicate GRB2/ALK protein complexes. Single ALK antibody staining was used as a control for PLA interactions. Scale bars, 10 μm. C, D. Bar graphs representing the number of PLA foci per cell from B, GRB2/ pALKY1604 PLA signals in H2228 cells and Beas2B V3. Data represent counts from at least 30 cells, n = 3. Error bar represents SD of three biological replicates. ****P < 0.0001 in comparison with DMSO (empty vector) by one-way ANOVA. Source data are available online for this figure. Download figure Download PowerPoint We next used a proximity ligation assay (PLA) to visualise pair-wise interactions between EML4-ALK V3 and GRB2 in the cytoplasm of Beas2B cells and H2228 and observed that the occurrence of these foci was significantly reduced in the presence of ALK inhibitors, ceritinib, alectinib and lorlatinib, as would be expected for a signalling complex mediated by SH2 domain binding to a tyrosine phosphorylated protein (Fig EV3B–D). These data confirm recruitment of the MAPK pathway adaptor protein, GRB2, to the cytoplasmic regions of EML4-ALK V3 foci and its loss upon ALK inhibition supporting the model that these foci potentially act as signalling hubs in the cytoplasm. To assess the impact of EML4-ALK V1 and V3 foci on these RAS/MAPK, PI3K/AKT and JAK/STAT pathways, we treated H3122 (V1) and H2228 (V3) cells with increasing doses of ALK inhibitors, ceritinib, alectinib and lorlatinib. In serum-starved H3122 cells, EML4-ALK V1 inhibition by ceritinib, alectinib or lorlatinib resulted in a dose-dependent loss of activated ALK (pY1604), ERK (pT202/Y204), AKT (pS473) and STAT3 (pY705) (Fig EV4A–C). Inhibition of EML4-ALK V3 in endogenous serum-starved H2228 cells using ceritinib and lorlatinib resulted in significant loss of ALK (pY1604), STAT3 (pY705) and ERK (pT202/Y204) phosphorylation but had minimal impact on AKT (pS473) (Fig EV4D and F). Notably, inhibition of EML4-ALK V3 with alectinib showed an increase of AKT (pS473) signal at most doses even when ALK (pY1604) activity was diminished (Fig EV4E). This suggests that H2228 (V3) cells are able to sustain RAS/MAPK and AKT/PI3K signalling pathways in the absence of ALK activity. Click here to expand this figure. Figure EV4. Effects of ALK inhibition on signalling pathways in H3122 and H2228 cells A–C. Representative Western blots of serum-starved H3122 cells treated with increasing doses of (A) ceritinib, (B) alectinib or (C) lorlatinib for 4 h. An equal volume of DMSO was used as control. Lysates were analysed by Western blotting and immunostaining for the proteins indicated and for their phosphorylation at the sites indicated. GAPDH was used as a loading control. Data represent of n = 2 experiments. D–F. Serum-starved H2228 cells were treated with increasing doses of (D) ceritinib, (E) alectinib or (F) lorlatinib for 4 h. Lysates were analysed by Western blotting and immunostaining with the indicated antibodies. GAPDH was used as a loading control. Data represent of n = 2 experiments. Source data are available online for this figure. Download figure Download PowerPoint Differential effects of ALK inhibitors on EML4-ALK localisation Targeting the EML4-ALK fusion in NSCLC patients with the TKIs, crizotinib, ceritinib and alectinib, has been broadly successful. However, cancers harbouring EML4-ALK V3 tend to respond less well to ALK inhibitors compared with cancers driven by EML4-ALK V1 (Kwak et al, 2010; Shaw et al, 2011; Solomon et al, 2014; Woo et al, 2017; Christopoulos et al, 2018). We therefore investigated the behaviour of EML4-ALK V1 and V3 cytoplasmic foci in the presence of ALK inhibitors. Consistent with the observation that catalytically inactive EML4-ALK V1 and V3 did not readily form cytoplasmic foci, incubation of HEK293 cells expressing WT EML4-ALK V1 and V3 with the ALK inhibitors, ceritinib and lorlatinib, led to loss of cytoplasmic foci with the V3 protein redirected to microtubules (Fig 3A and B, E and F). Meanwhile, ceritinib and lorlatinib treatment led to further enrichment of the V3 KD (kinase-dead) protein on microtubules (Fig 3D and H), with V1 KD remaining widely dispersed in the cytoplasm (Fig 3C and G). Figure 3. Different ALK inhibitors dissolve or maintain cytoplasmic EML4-ALK V1 and V3 foci and redirect V3 to microtubules A–D. HEK293 cells were transfected with EML4-ALK V1 WT or V3 WT or kinase dead (KD) for 48 h. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 h before fixation and staining with anti-GFP (green), anti-α-tubulin (red) and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. E, F. Violin plo