Extended Data Fig. 4: Kinobead-based proteomic identification of the trametinib and nintedanib targets and treatment-induced changes in the phosphoproteome of classical and mesenchymal PDAC.

a, b, Representative pictures of the target space of trametinib (a) and nintedanib (b) for 2259 PDAC cells. A phylogenetic tree of all kinases for the 2259 primary mouse PDAC cell culture is shown. The indicated circle sizes indicate potency (apparent dissociation constants (Kdapp)), the color code specifies protein-drug interaction with the designated or other targets. Arrows highlight the identified targets. c, Radar plot showing the overlay of the pKd (−log10Kd) for the targets of nintedanib in the 6 PDAC cell cultures tested. PDAC cells of the classical (n = 4) and mesenchymal (n = 2) subtype are indicated with the color code. d, Heatmap showing the differentially expressed genes between epithelial and mesenchymal cell cultures identified as targets of nintedanib. The color code indicates the Z score. e, Volcano plots representing the change in gene expression of the nintedanib targets (in blue) upon trametinib treatment. The x-axis log2 fold change (treated/control), the y-axis shows the per test adjusted p values, which were calculated by differential expression test (using the DESeq2 package). A gene was considered to be differentially expressed with a Benjamini-Hochberg adjusted p-value of 0.05 and an absolute fold change >1. f, g, Mesenchymal (9091) and classical (8661) PDAC cell cultures were used to generate site-specific phospho-array datasets (Phospho Explorer antibody microarray, Full Moon Biosystems). The cell lines were analyzed at basal condition and in presence of T/N (trametinib 10 nM + nintedanib 2 μM). Phospho-array data (Supplementary Table 3) were used to test for the decrease of differentially phosphorylated sites between T/N and vehicle (DMSO) treated classical and mesenchymal mPDAC cells. Functionally grouped maps, obtained with the ClueGO plugin of Cytoscape, representing pathways overrepresented in mesenchymal (f) and classical (g) mPDAC upon T/N treatment are shown. Only the pathways with an adjusted p value (calculated by two-sided hypergeometric test, Bonferroni corrected) ≤ 0.05 are represented. The node size represents the term enrichment significance.