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

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)-triggered by radiation-induced double strand breaks-is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

• MeSH
• Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors   MeSH
• Phosphorylation drug effects   radiation effects   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Proteome metabolism   MeSH
• Pyrazines pharmacology   MeSH
• Radiation-Sensitizing Agents pharmacology   MeSH
• Sulfones pharmacology   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
• Protein Kinase Inhibitors MeSH
• Proteome MeSH
• Pyrazines MeSH
• Radiation-Sensitizing Agents MeSH
• Sulfones MeSH

We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 μM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 μM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 μM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 μM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.

• MeSH
• Leukemia, Promyelocytic, Acute pathology   MeSH
• Apoptosis drug effects   MeSH
• Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors   MeSH
• HL-60 Cells MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• G2 Phase Cell Cycle Checkpoints drug effects   MeSH
• Humans MeSH
• Morpholines pharmacology   MeSH
• DNA Repair drug effects   MeSH
• Pyrazines pharmacology   MeSH
• Pyrones pharmacology   MeSH
• Sulfones pharmacology   MeSH
• Radiation Tolerance drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one MeSH Browser
• 3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR protein, human MeSH Browser
• Protein Kinase Inhibitors MeSH
• Morpholines MeSH
• Pyrazines MeSH
• Pyrones MeSH
• Sulfones MeSH