Fig. 2: Nuclear localization and function of YAP in DTPs.

a Schematic representation of YAP nuclear localization and interaction with transcription factors in DTPs. Created with BioRender.com. b, c YAP levels in nuclear lysates evaluated across PC9 and H1975 cells treated with osimertinib (Osim.), H3122 and H2228 cells treated with alectinib (Alec.), as well as H358 cells and H1838 cells treated with SHP2 inhibitor RMC-4550. Lysates were collected at indicated time points. For PC9 cells, a detailed time course is presented, including corresponding osimertinib-resistant PC9-AR. n = 3 independent experiments. d PanTEAD-YAP proximity ligation assay (PLA) in PC9 cells treated with osimertinib, analyzed by confocal microscopy. Image is representative of 200 or more cells per condition in total n = 4 independent experiments. e, f Quantification of percentage of cells from (d), mean ± s.d., two-sided t test, ***p = 1.08 x 10^-4 (DMSO vs. Osim.-2d), ***p = 7.32 x 10^-21 (DMSO vs. Osim.-9d), **p = 0.0023 (DMSO vs. washout), **p = 2.35 x 10^-43 (Osim.-9d vs. washout). g, h YAP target genes were significantly induced in PC9 and H3122 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test, (g) **p = 0.0028, (h) **p = 0.0018, ***p < 0.0001. i, j YAP target genes were significantly induced in DTP-specific states versus acquired-resistant states in PC9 and H3122 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test, (i) **p = 0.0031, (j) **p = 0.0002, (i, j) ***p < 0.0001. k Significant decrease in relative DTP cell numbers upon siRNA-mediated YAP knockdown during DTP development in PC9 and H3122 cells. n = 3 independent experiments, mean ± s.d., two-sided t test. l Significant decrease in relative cell numbers upon siRNA-mediated YAP knockdown in PC9 and H2228 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test. m Cell viability of PC9 DTPs was significantly decreased by treatment with TEAD inhibitor VT-104 (TEADi). n = 3 independent experiments, mean ± s.d., two-sided t test, **p = 0.0024, **p = 0.0023, **p = 0.0026, ***p < 0.0001. n Compared to parental or AR cells, combined treatment with osimertinib and TEADi exerted a significant effect and reduced cell viability, specifically in PC9 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test, **p = 0.0024, **p = 0.0023, *p = 0.0198, ***p < 0.0001. o Gene set enrichment analysis (GSEA) for the YAP-5SA_UP gene set (Supplementary Data 3) using RNAseq expression data from untreated parental control compared to PC9 DTPs, H3122 DTPs and H358 DTPs. NES, Nominal Enrichment Score; FDR, False Discovery Rate.