biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsMira BurtscherCoulter Automated Workstation Biomek i7 HybridnoneNextSeq 2000RNeasy 96-well plate kitRNA-seq of coding RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16292Dysregulated kinase activity drives oncogenic signalling, perturbs cellular homeostasis, and promotes tumour progression. Despite major success in targeting kinases therapeutically, the downstream consequences of kinase inhibition and the mechanisms underlying drug resistance remain incompletely understood. One of the most frequent oncogenic kinase mutations, BRAFV600E, constitutively activates the MAPK pathway and represents a major therapeutic target in melanoma and other cancers. However, the functional relevance of most phosphorylation events downstream of BRAF signalling is unknown, limiting mechanistic interpretation and rational therapeutic design. Here, we established a global, multi-omic model of BRAF inhibition response in BRAFV600E-mutant melanoma cells by integrating time-resolved phosphoproteomics, biophysical PTM-proteomics, transcriptomics, and thermal proteome profiling. Our ultradeep phosphoproteomic analysis revealed widespread phosphorylation changes upon Dabrafenib treatment, while biophysical phosphoproteomics uncovered phosphorylation events associated with altered solubility and subcellular localisation, indicative of biomolecular condensation and nuclear reorganisation. Integration of these modalities into a network-based mechanistic model enabled the prioritisation of functionally relevant phosphorylation sites and kinases. Experimental validation confirmed CDK9, CLK3, and TNIK as key regulators of BRAFV600E signalling and as candidate targets for combinatorial inhibition strategies capable of re-sensitising resistant melanoma cells in a synthetic lethal manner. The transcription factor ETV3 emerged from the network as a previously unrecognised effector of oncogenic BRAF signalling. Using phosphosite-specific biophysical data, imaging, and FRAP experiments, we demonstrated that ETV3 phosphorylation controls its DNA-binding kinetics. Functional assays combining ETV3 knockdown, metabolomics, and drug screening revealed that ETV3 modulates transcriptional and metabolic responses to BRAF inhibition, linking oncogenic signalling to metabolic rewiring. Together, this study provides a comprehensive systems-level framework that connects phosphorylation dynamics to protein function and cellular phenotype, highlights ETV3 as a novel signalling node, and illustrates how multi-omic, site-resolved network models can reveal actionable mechanisms of kinase-driven oncogenesis.biostudies-arrayexpressSample Collection - Approximately 8*10^6 cells were lysed in 600 µl RLT buffer (Qiagen, #74181) and RNA was extracted using the RNeasy 96-well plate kit (Qiagen, #74181) according to manufacturer’s instructions including the DNase step.Nucleic Acid Extraction - Approximately 8*10^6 cells were lysed in 600 µl RLT buffer (Qiagen, #74181) and RNA was extracted using the RNeasy 96-well plate kit (Qiagen, #74181) according to manufacturer’s instructions including the DNase step.Sequencing - The pool was loaded and sequenced on an Illumina NextSeq 2000 platform (Illumina, San Diego, CA, USA) using a P3 100 cycle kit, a read-length of 122bp single-end reads and 650pM final loading concentrationLibrary Construction - The initial RNA was QCed using Agilent Bioanalyzer with the RNA Nano Assay kit as per the manufacturer’s protocol. The RNA sample set was then standardized to 200 ng total RNA in 50 ul using the concentration values given by the Bioanalyzer. The libraries were prepared on a Beckman Coulter Automated Workstation Biomek i7 Hybrid (MC + Span-8). For library preparation an automated version of the NEBNext® Ultra™ II Directional RNA Library Prep Kit was used, following section 1 - Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module 60. An adaptor dilution of 1 to 30 was used, the samples were individually barcoded using unique dual indices during the PCR using 12 PCR cycles as per the manufacturer’s protocol. The individual libraries were quantified using the Qubit HS DNA assay as per the manufacturer’s protocol. For the measurement 1ul of sample in 199ul of Qubit working solution was used. The quality and molarity of the libraries was assessed using Agilent Bioanalyzer with the DNA HS Assay kit as per the manufacturer’s protocol[v]. The assessed molarity was used to equimolarly combine the individual libraries into one pool for sequencing.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesMira BurtscherfalseCharting the functional landscape of phosphorylation through biophysical and multi-omics integrationDysregulated kinase activity drives oncogenic signalling, perturbs cellular homeostasis, and promotes tumour progression. Despite major success in targeting kinases therapeutically, the downstream consequences of kinase inhibition and the mechanisms underlying drug resistance remain incompletely understood. One of the most frequent oncogenic kinase mutations, BRAFV600E, constitutively activates the MAPK pathway and represents a major therapeutic target in melanoma and other cancers. However, the functional relevance of most phosphorylation events downstream of BRAF signalling is unknown, limiting mechanistic interpretation and rational therapeutic design. Here, we established a global, multi-omic model of BRAF inhibition response in BRAFV600E-mutant melanoma cells by integrating time-resolved phosphoproteomics, biophysical PTM-proteomics, transcriptomics, and thermal proteome profiling. Our ultradeep phosphoproteomic analysis revealed widespread phosphorylation changes upon Dabrafenib treatment, while biophysical phosphoproteomics uncovered phosphorylation events associated with altered solubility and subcellular localisation, indicative of biomolecular condensation and nuclear reorganisation. Integration of these modalities into a network-based mechanistic model enabled the prioritisation of functionally relevant phosphorylation sites and kinases. Experimental validation confirmed CDK9, CLK3, and TNIK as key regulators of BRAFV600E signalling and as candidate targets for combinatorial inhibition strategies capable of re-sensitising resistant melanoma cells in a synthetic lethal manner. The transcription factor ETV3 emerged from the network as a previously unrecognised effector of oncogenic BRAF signalling. Using phosphosite-specific biophysical data, imaging, and FRAP experiments, we demonstrated that ETV3 phosphorylation controls its DNA-binding kinetics. Functional assays combining ETV3 knockdown, metabolomics, and drug screening revealed that ETV3 modulates transcriptional and metabolic responses to BRAF inhibition, linking oncogenic signalling to metabolic rewiring. Together, this study provides a comprehensive systems-level framework that connects phosphorylation dynamics to protein function and cellular phenotype, highlights ETV3 as a novel signalling node, and illustrates how multi-omic, site-resolved network models can reveal actionable mechanisms of kinase-driven oncogenesis.2026-01-01T00:00:00Z2026-01-02T02:03:15.439Z2025-11-25T17:54:31.182ZE-MTAB-16292EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184