Study on Radiosensitization of Human Leukemic Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis

DNA damage response (DDR) is an essential mechanism to maintain genomic stability as its activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in resistance towards various genotoxic insults. Radiation-induced DDR is regulated mainly by three phosphatidylinositol 3-kinase-related kinases (PIKKs): ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and ATM and Rad3-related kinase (ATR). Therefore, the therapeutic inhibition of PIKKs represents a promising strategy to increase radiosensitivity in cancer cells.

In the presented chapter, we aimed to elucidate molecular mechanisms underlying the radiosensitization of leukemic cells by PIKKs inhibitors. We focused on recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), that has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. We evaluated the impact of VE-821 on proliferation, viability, and cell cycle of sham-irradiated and irradiated leukemic cells and compared it to the effect of another PIKK inhibitor KU55933, targeting ATM. Both inhibitors proved to radiosensitize MOLT-4 cells, and additionally, 10 µM VE-821 was shown to act as an effective antiproliferative agent in sham-irradiated human leukemic T-lymphocyte MOLT-4 cells.

Furthermore, we employed SILAC-based quantitative phosphoproteomics to describe in detail 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 identified 9834 phosphorylation sites. Further analysis revealed 328 differentially regulated phosphoproteins (false discovery rate 1%).

Proteins with up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in all phases of the cell cycle and cell division. Pre-incubation with VE-821 combined with IR modulated the mechanisms of the G1/S transition, impacted the intra-S-checkpoint, disrupted the G2/M checkpoint, and altered the activity of kinases involved in mitosis. In addition, we found that ATR inhibition impacted phosphoproteins involved in DDR, gene expression, and apoptosis. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes.

In conclusion, our phosphoproteomic analyses allowed us to provide a comprehensive description of ATR kinase inhibition and its impact on cellular response to IR-induced genotoxic stress. Taken together, our data indicate that ATR has multiple roles in response to DNA damage and that its inhibitor VE-821 is a potent radiosensitizing agent for leukemic cells.