Fig. 2: EML4-ALK suppresses EGFR signaling.
From: Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance
A EGF stimulates EGFR and downstream RAS/ERK signaling. B ppERK levels in response to EGF (100 ng/mL) in the presence of 1 µM ALKi (orange) or DMSO (gray) in STE-1 and H3122 cancer cells. Data points represent mean of 1000–2800 cells for STE-1 and 800–3000 cells for H3122. Error bars = 95% CI. ****p < 0.0001, ***p = 0.0007, *p = 0.02 by one-sided T-test comparing ALKi vs DMSO. n = 3 biological replicates. C Representative images of ALKi-dependent potentiation of ERK response to EGF (B). D Fold-change increase over a range of EGF concentrations. Data represent ratio of mean ppERK from EGF-stimulated (15 min) vs unstimulated cells. Significance assessed using one-sided T-test, n = 3 biological replicates. E, F Representative single-cell (top) and average (bottom) ERK responses to EGF (50 ng/mL, 15 min) and ALKi (crizotinib, 1 μm, 2 h pretreatment) in two primary patient-derived cell lines, CUTO-8 and CUTO-9, that harbor EML4-ALK(V1) (E), or in cell lines driven by a constitutively active, full-length ALK mutant (F). Data points in (E, F) (bottom) represent mean ppERK normalized to mean ppERK of ALKi-treated cells for 900–2100 STE-1, 900–1500 CUTO-8, 200–300 CUTO-9, 200–600 Kelly cells and 1700–2700 SY5Y cells. Significance assessed by one-sided T-test, n = 3 biological replicates for STE-1, CUTO-8, Kelly, and SY5Y; n = 6 biological replicates for CUTO-9. G EML4-ALK(V1) (EML4-ALK-2A-H2B-miRFP) or a control construct (H2B-miRFP) were transiently expressed in lung epithelial Beas2B cells. H Time course of ppERK immunofluorescence levels in response to EGF stimulation (50 ng/mL). Data points represent mean ± SEM of 120–300 cells for untransfected Beas2B and 80–160 cells for Beas2B expressing EML4-ALK. **p = 0.003, *p = 0.04, by one-sided T-test for ppERK in transfected vs untransfected cells, n = 3 biological replicates. I Dynamic range of ppERK in EML4-ALK-expressing Beas2B in response to EGF in the presence or absence of ALKi (1 µM) pretreatment. Significance assessed using one-sided T-test, n = 3 biological replicates.
