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Abstract
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.
Highlights
One of the treatment modalities in oncology is radiotherapy
We aimed to characterize the effect of specific inhibition of ATR kinase by VE-821 and to report the mechanisms and signaling pathways involved in the processes triggered by ionizing radiation (IR) in p53-negative human promyelocytic leukemic cells HL-60 that are ATR-dependent
We employed SILAC-based quantitative phosphoproteomics together with metal oxide affinity chromatography using TiO2 microparticles to enrich for phosphorylated peptides
Summary
One of the treatment modalities in oncology is radiotherapy. It often employs chemical agents increasing sensitivity towards ionizing radiation (IR). IR induces the most deleterious lesions of DNA, double strand breaks (DSB), and their repair is regulated by ataxia telangiectasia-mutated kinase (ATM), DNA-dependent protein kinase (DNA-PK), and ATM and Rad3-related kinase (ATR). While activation of ATM and DNA-PK is triggered by DNA double stranded breaks, ATR kinase responds to a broad spectrum of agents inducing single stranded DNA [1]. ATR acts primarily in Sand G2-phases and responds to replication and genotoxic stress, a recent report has shown that ATR is activated in irradiated G1 phase cells [2]. VE-821 selectivity is based on the concept that (i) more than 50% of cancer cells have lost their G1-phase checkpoint for example due to p53 mutation/deletion and rely on S- and G2-checkpoints; which are known to be regulated by ATR and (ii) cancer cells with activated oncogenes generate replication stress at much higher levels than normal cells, activating
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