Study on Radiosensitization of Human Leukemic Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis | Chapter 8 | New Ideas Concerning Science and Technology Vol. 9

The DNA damage response (DDR) is a critical mechanism for maintaining genomic stability since it promotes cell-cycle arrest and encourages DNA damage repair, resulting in resistance to a variety of genotoxic insults when activated. Ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and ATM and Rad3-related kinase are the three phosphatidylinositol 3-kinase-related kinases (PIKKs) that control radiation-induced DDR (ATR). As a result, inhibiting PIKKs therapeutically may be a promising technique for increasing radiosensitivity in cancer cells. The aim of this chapter was to uncover the molecular mechanisms underlying PIKKs inhibitors' radiosensitization of leukemic cells. We concentrated on VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), a recently developed specific ATR inhibitor that has been documented to have an important radio- and chemo-sensitizing effect limited to cancer cells (mostly p53-deficient) without affecting normal cells. We compared the effects of VE-821 on leukemic cell proliferation, viability, and cell cycle in sham-irradiated and irradiated cells to the effect of another PIKK inhibitor, KU55933, which targets ATM. MOLT-4 cells were radiosensitized by both inhibitors. In addition, we used SILAC-based quantitative phosphoproteomics to explain the mechanism of VE-821's radiosensitizing effect in human promyelocytic leukemic cells HL-60 in detail (p53-negative). 9834 phosphorylation sites were discovered using hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography – tandem mass spectrometry (LC-MS/MS) study. Further investigation found 328 phosphoproteins that were differentially regulated (false discovery rate 1 percent ). Proteins whose phosphorylation was up- or down-regulated were mainly found in the nucleus and were involved in all phases of the cell cycle and cell division. The effects of VE-821 combined with IR on the mechanisms of the G1/S transition, the intra-S-checkpoint, the G2/M checkpoint, and the function of mitosis-related kinases were all altered. Furthermore, we discovered that inhibiting ATR had an effect on phosphoproteins involved in DDR, gene expression, and apoptosis. Furthermore, sequence motif analysis showed that the behaviours of kinases involved in these processes had changed significantly. Finally, our phosphoproteomic studies allowed us to provide a detailed explanation of ATR kinase inhibition and its effect on cellular response to IR-induced genotoxic stress. Our findings show that ATR plays multiple roles in the response to DNA damage, and that its inhibitor VE-821 is an effective radiosensitizer for leukemic cells.

Author (s) Details
 
Barbora Šalovská
Department of Radiobiology, Faculty of Military Health Sciences in Hradec Králové, University of Defense in Brno, Hradec Kralove, 500 01, Czech Republic.

Aleš Tichý
Department of Radiobiology, Faculty of Military Health Sciences in Hradec Králové, University of Defense in Brno, Hradec Kralove, 500 01, Czech Republic.

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