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.

• MeSH
• Amino Acid Motifs MeSH
• Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors   MeSH
• Biomarkers MeSH
• Phosphoproteins * chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Gene Ontology MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Cell Cycle Checkpoints drug effects   radiation effects   MeSH
• Humans MeSH
• Metabolome * MeSH
• Metabolomics methods   MeSH
• Cell Line, Tumor MeSH
• Proteome * MeSH
• Proteomics methods   MeSH
• Pyrazines pharmacology   MeSH
• Radiation-Sensitizing Agents pharmacology   MeSH
• Signal Transduction MeSH
• Sulfones pharmacology   MeSH
• Radiation Tolerance drug effects   MeSH
• TOR Serine-Threonine Kinases metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Computational Biology methods   MeSH
• Gamma Rays MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR protein, human MeSH Browser
• Biomarkers MeSH
• Phosphoproteins * MeSH
• Protein Kinase Inhibitors MeSH
• MTOR protein, human MeSH Browser
• Proteome * MeSH
• Pyrazines MeSH
• Radiation-Sensitizing Agents MeSH
• Sulfones MeSH
• TOR Serine-Threonine Kinases MeSH