Fig. 3: Upstream regulation of YAP by FAK signaling in DTPs.

a Changes in the FAK expression signature30 in PC9 cells treated with 2 μM osimertinib, H3122 cells treated with 500 nM alectinib, and H358 cells treated with 10 μM RMC-4550. Statistical significance is indicated by two-way ANOVA test, n = 3 independent experiments. The box plots display 25th (lower bound), 50th (centre, median), and 75th (upper bound) percentiles, with whiskers (minima (bottom), maxima (top)) extending 1.5 * IQR. b Actin cytoskeleton changes upon treatment with 2 μM osimertinib in PC9 and H1975 cells. Image is representative in total n = 3 independent experiments, scale bar: 10 μm. c Phosphorylation changes of key FAK signaling molecules including EphB1, ACK1, and FAK, as well as for the YAP activating Y357 and inactivating S127 phosphorylation site upon treatment with targeted inhibitors in PC9 cells and H3122 cells. n = 3 independent experiments. d The relative number of DTPs decreased upon combinatorial knockdown of EphB1, ACK1, and FAK in PC9 DTPs and H3122 DTPs compared to non-target control (siNT). n = 3 independent experiments, mean ± s.d., two-sided t test. e Decrease in total YAP expression and nuclear localization upon simultaneous knockdown of EphB1, ACK1, and FAK in PC9 DTPs and H3122 DTPs. n = 3 independent experiments. f The relative number of PC9 osimertinib (2uM) DTPs and H358 RMC-4550 (10uM) DTPs was reduced in cells harboring a CRISPR-mediated FAK or YAP knockout (KO). n = 3 independent experiments, mean ± s.d., two sided t test. g Changes in YAP expression and nuclear localization upon FAK-KO in parental PC9 cells. n = 3 independent experiments. h Knockdown of FAK decreased nuclear YAP expression levels in PC9 DTPs. n = 3 independent experiments. i Normalized DTP numbers upon targeted therapies in combination with FAK inhibitor VS-4718 across PC9 DTPs, H3122 DTPs, and H358 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test. j Decrease in YAP nuclear localization in PC9 cells (day 5) and H3122 cells (day 2) upon combined treatment with FAK inhibitor VS-4718 and the backbone targeted treatment; scale bar: 10 μm. Quantification of relative integrated density was performed by automated analysis quantifying the intensity for the protein of interest per nuclei. Image is representative in total n=3 independent experiments, two-sided t-test.