Radio-sensitizing effects of VE-821 and beyond: Distinct phosphoproteomic and metabolomic changes after ATR inhibition in irradiated MOLT-4 cells

• Published in: PloS one Vol. 13; no. 7; p. e0199349
• Main Authors: Šalovská, Barbora, Janečková, Hana, Fabrik, Ivo, Karlíková, Radana, Čecháková, Lucie, Ondrej, Martin, Link, Marek, Friedecký, David, Tichý, Aleš
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 12-07-2018; Public Library of Science (PLoS)
• Subjects: Abnormalities; Amino Acid Motifs; Analysis; Ataxia Telangiectasia Mutated Proteins - antagonists & inhibitors; Binding Sites; Biochemistry; Biology; Biology and Life Sciences; Biomarkers; Biomedical research; Cancer; Cancer therapies; Cell culture; Cell Cycle Checkpoints - drug effects; Cell Cycle Checkpoints - radiation effects; Cell Line, Tumor; Cell proliferation; Cells (Biology); Chemotherapy; CHK1 protein; Computational Biology - methods; Dentistry; Deoxyribonucleic acid; DNA; DNA damage; DNA repair; Gamma Rays; Gene expression; Gene Ontology; Health sciences; Humans; Inhibition; Inhibitors; Ionizing radiation; Irradiated; Irradiation; Kinases; Laboratory animals; Leukemia; Lymphocytes T; Medicine; Metabolic disorders; Metabolism; Metabolites; Metabolome; Metabolomics; Metabolomics - methods; Molecular chains; Molecular modelling; Molting; Mutation; Oxidative stress; p53 Protein; Pathways; Phenotypes; Phosphoproteins - chemistry; Phosphoproteins - metabolism; Phosphorylation; Protein Binding; Protein Kinase Inhibitors - pharmacology; Proteome; Proteomics; Proteomics - methods; Pyrazines - pharmacology; Radiation; Radiation damage; Radiation Tolerance - drug effects; Radiation-Sensitizing Agents - pharmacology; Radiosensitization; Research and Analysis Methods; Sensitizing; Signal Transduction; Studies; Sulfones - pharmacology; TOR protein; TOR Serine-Threonine Kinases - metabolism; Tumors; United States > US; Czech Republic
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0199349

Current anti-cancer strategy takes advantage of tumour specific abnormalities in DNA damage response to radio- or chemo-therapy. Inhibition of the ATR/Chk1 pathway has been shown to be synthetically lethal in cells with high levels of oncogene-induced replication stress and in p53- or ATM- deficient cells. In the presented study, we aimed to elucidate molecular mechanisms underlying radiosensitization of T-lymphocyte leukemic MOLT-4 cells by VE-821, a higly potent and specific inhibitor of ATR. We combined multiple approaches: cell biology techniques to reveal the inhibitor-induced phenotypes, and quantitative proteomics, phosphoproteomics, and metabolomics to comprehensively describe drug-induced changes in irradiated cells. VE-821 radiosensitized MOLT-4 cells, and furthermore 10 μM VE-821 significantly affected proliferation of sham-irradiated MOLT-4 cells. We detected 623 differentially regulated phosphorylation sites. We revealed changes not only in DDR-related pathways and kinases, but also in pathways and kinases involved in maintaining cellular metabolism. Notably, we found downregulation of mTOR, the main regulator of cellular metabolism, which was most likely caused by an off-target effect of the inhibitor, and we propose that mTOR inhibition could be one of the factors contributing to the phenotype observed after treating MOLT-4 cells with 10 μM VE-821. In the metabolomic analysis, 206 intermediary metabolites were detected. The data indicated that VE-821 potentiated metabolic disruption induced by irradiation and affected the response to irradiation-induced oxidative stress. Upon irradiation, recovery of damaged deoxynucleotides might be affected by VE-821, hampering DNA repair by their deficiency. Taken together, this is the first study describing a complex scenario of cellular events that might be ATR-dependent or triggered by ATR inhibition in irradiated MOLT-4 cells. Data are available via ProteomeXchange with identifier PXD008925.