Fig 2: PDGFR and SRC kinase co-expression in high-grade gliomas. (A) Co-expression analysis of the indicated genes from the TCGA Glioblastoma dataset. (B) Overall expression of pSKF (using a phospho-SRC antibody), PDGFRA and PDGFRB in a TMA containing 70 stage 4 Glioblastoma and 10 healthy cerebrum tissue samples. (C) Quantification of relative PDGFRA expression in TMA GBM samples; scale bar, 50 µm. (D) Quantification of relative PDGFRB expression in TMA GBM samples; scale bar, 50 µm. (E) Quantification of relative pSFK expression in TMA GBM samples; scale bar, 50 µm. (F) Quantification of relative expression of PDGFRA and PDGFRB in high pSFK tumour samples.

Fig 3: Dexamethasone induces PDGFR mediated survival signalling. (A) Functional characteristics of T98G cells treated with 25 µM DEXA for 18 h revealed by GSEA using GO term and PID gene set collections. (B) qRT-PCR analysis for PDGFRA, PDGFRB and PDGFB in the indicated cell lines represented as mean fold change of triplicates treated with DEXA for 18 h relative to DMSO. (C) Western blot analysis for PDGFRA and PDGFRB in the indicated cell lines either untreated or treated with DEXA for 18 h. Beta-actin served as loading control. (D) Western blot analysis for PDGFRA, PDGFRB and the indicated phospho-specific antibodies in T98G cells either untreated or treated with DEXA for 18 h. The lysate of one experiment was run on several lanes per blot, which were probed in parallel. Beta-actin serves as loading control and representative controls are shown. (E) Western blot analysis for phospho-SFK (using a phospho SRC antibody) and phospho-STAT5 in the indicated cell lines either untreated or treated with DEXA for 18 h. Beta-actin served as loading control. (F) Co-expression analysis of the indicated genes from the TCGA Glioblastoma dataset. (G) qRT-PCR analysis for BCL2L1 and MCL1 in the indicated cell lines represented as mean fold change of triplicates treated with DEXA for 18 h relative to DMSO. (H) qRT-PCR analysis as in (G). Cells were treated with DEXA in the absence or presence of 200 nM ponatinib for 18 h. (I) Quantification of the relative cell number of the indicated cell lines grown in the absence or presence of 25 µM DEXA. Data represent the mean ± SEM (n = 3). (J) Quantification of the relative cell number of the indicated cell lines grown with or without 25 µM DEXA in the absence or presence of 25 nM or 100 nM ponatinib (Pon) or 1 µM or 3 µM sunitinib (Sun). Data represent mean ± SEM (n = 3). (K) Quantification of the relative cell number of T98G cells grown with or without 25 µM DEXA in the absence or presence of 5 µM S63845 (S63), 1 µM navitoclax (Nav) or 5 µM venetoclax (Ven). Data represent mean ± SEM (n = 3). ** p < 0.01, *** p < 0.001.

Fig 4: Mapping the distinct kinome signatures among GIST subtypes.(A–C) PCA, including PC1 vs. PC2 (A), PC1 vs.PC3 (B), and PC2 vs.PC3 (C) of MIB-MS in 3 GIST subtypes (KIT mutant, blue; PDGFRA mutant, red; WT, green) and normal gastric tissue (pink). (D) Volcano plot comparisons of KIT mutant vs. WT, (E) PDGFRA mutant vs. WT, and (F) KIT mutant vs. PDGFRA mutant GIST MIB-MS kinome profiles. Differences in kinase log2 LFQ intensities among tumors and normal tissues determined by paired t test Benjamini-Hochberg adjusted P values at FDR of <0.05 using Perseus software. PCA, principal component analysis; GIST, gastrointestinal stromal tumor; MIB-MS, multiplexed inhibitor beads and mass spectrometry; LFQ, label-free quantitation.

Fig 5: Characterizing the GIST kinome in primary tumors using MIB-MS to identify therapeutic targets.(A) Schematic of experimental approach. MIB-MS was used to quantify the kinase abundance in patients with GISTs (untreated, gastric primary GIST from 3 molecular subtypes: KIT mutant, n = 15; PDGFRA mutant, n = 10; WT GIST, n = 8; and normal gastric tissue, n = 9) to map the proteomic landscape of the kinome and identify targets. Kinase levels in tissues were determined using a combination of LFQ and s-SILAC. (B) Kinome tree depicts fraction of kinome quantitated by MIB-MS and frequency across 42 samples measured. (C) Average number of kinases detected by MIB-MS profiling broken down by tissue type. GIST, gastrointestinal stromal tumor; MIB-MS, multiplexed inhibitor beads and mass spectrometry; LFQ, label-free quantitation; s-SILAC, super-SILAC.