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ATR inhibition rewires cellular signaling networks in response to pathologic replication stress

ABSTRACT: DNA replication stress is the major cause of genomic instability in human cells. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancer. However, the ATR-dependent signaling in the cellular response to replication stress has not been systematically investigated. Here, we employ quantitative mass spectrometry-based proteomics to decipher the ATR-dependent phosphorylation events in response to pathological replication stress induced by hydroxyurea. Our data define the substrate spectrum of ATR and identify several novel putative ATR substrates. In addition, we demonstrate that ATR-inhibition fundamentally rewires cellular signaling networks leading to the prominent activation of different pathways including the ATM-driven double strand break repair.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Petra Beli

PROVIDER: MSV000080795 | MassIVE | Wed Mar 29 16:38:00 BST 2017

SECONDARY ACCESSION(S): PXD002135

REPOSITORIES: MassIVE

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ATR inhibition rewires cellular signaling networks induced by replication stress.

Wagner Sebastian A SA Oehler Hannah H Voigt Andrea A Dalic Denis D Freiwald Anja A Serve Hubert H Beli Petra P

Proteomics 20160201 3

The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling ...[more]

PMID: 26572502

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Project description:The cell division cycle is tightly controlled in normal cells. Cancer cells are characterized by deregulation in cell division cycle, which is associated with increased DNA replication and elevated cellular proliferation. Recently, genetic studies indicate that cyclin dependent kinases CDK2, CDK4 and CDK6 are not essential for the mammalian cell cycle, instead, they are only required for the proliferation of specific cell types.Thus, targeting the activities of CDK2, CDK4 and CDK6 may be a promising approach for cancer treatment. Rocaglamides (Roc) (= Flavaglines), derived from the traditional Chinese medicinal plant Aglaia, are a group of naturally occurring herbal chemicals characterized by a cyclopenta[b]benzofurans skeleton. A number of Roc derivatives have been found to have potent inhibitory effects on tumor growth with IC50 concentrations at the nM rages. Roc-mediated inhibition of proliferation was first found to be associated with inhibition of protein synthesis in 1998. In this study, using the representative Roc-A compound we revealed a novel mechanistic function of Roc. We show that Roc-A induces rapid activation of ATM (ataxiatelangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) kinases. Activated ATM and ATR in turn activate the downstream checkpoint kinases CHK1 and CHK2. The latter then phosphorylate Cdc25A that leads to a phosphorylation-mediated degradation of Cdc25A. Usually, ATR and ATM are activated in response to DNA damage. However, in this study we show that Roc-A does not directly induce DNA breaks. To elucidate the molecular mechanism by which Roc-A activates ATM and ATR we performed a time-resolved microarray analysis to compare Roc-A-inducible genes in malignant vs. normal T lymphocytes. Based on the dynamic gene responses we found that Roc-A stimulates in Jurkat, but not in normal T lymphocytes, a set of genes responsive to DNA damage and nucleotide excision repair. This finding provides a hint of the molecular bases for Roc-induced activation of ATM and ATR. This finding also revealed a new scope for cancer treatment using Roc-A. The isolation time points for Roc A treated D6 T-cells were recorded at t=[1,2,3,4,6,8,10,14,20,28,36] hours after stimulation. Post-stimulation time points for Jurkat cells are t=[0.5,1,2,3,4,5,6,7,8,10,12,14,16,20,24,28,32,36]. Unstimulated control time points were taken at t=[0,4,10,16,24,36] hours for Jurkat cells at t=[0,6,14,24,36] hours.