A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations.
ABSTRACT Oncogene activation has been shown to generate replication-born DNA damage, also known as replicative stress. The primary responder to replicative stress is not Ataxia-Telangiectasia Mutated (ATM) but rather the kinase ATM and Rad3-related (ATR). One limitation for the study of ATR is the lack of potent inhibitors. We here describe a cell-based screening strategy that has allowed us to identify compounds with ATR inhibitory activity in the nanomolar range. Pharmacological inhibition of ATR generates replicative stress, leading to chromosomal breakage in the presence of conditions that stall replication forks. Moreover, ATR inhibition is particularly toxic for p53-deficient cells, this toxicity being exacerbated by replicative stress-generating conditions such as the overexpression of cyclin E. Notably, one of the compounds we identified is NVP-BEZ235, a dual phosphatidylinositol-3-OH kinase (PI3K) and mTOR inhibitor that is being tested for cancer chemotherapy but that we now show is also very potent against ATM, ATR and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).
SourceAvailable from: Miiko Sokka[Show abstract] [Hide abstract]
ABSTRACT: Nucleoli are not only organelles that produce ribosomal subunits. They are also overarching sensors of different stress conditions and they control specific nucleolar stress pathways leading to stabilization of p53. During DNA replication, ATR and its activator TopBP1 initiate DNA damage response upon DNA damage and replication stress. We found that a basal level of TopBP1 protein associates with ribosomal DNA repeat. When upregulated, TopBP1 concentrates at the ribosomal chromatin and initiates segregation of nucleolar components-the hallmark of nucleolar stress response. TopBP1-induced nucleolar segregation is coupled to shut-down of ribosomal RNA transcription in an ATR-dependent manner. Nucleolar segregation induced by TopBP1 leads to a moderate elevation of p53 protein levels and to localization of activated p53 to nucleolar caps containing TopBP1, UBF and RNA polymerase I. Our findings demonstrate that TopBP1 and ATR are able to inhibit the synthesis of rRNA and to activate nucleolar stress pathway; yet the p53-mediated cell cycle arrest is thwarted in cells expressing high levels of TopBP1. We suggest that inhibition of rRNA transcription by different stress regulators is a general mechanism for cells to initiate nucleolar stress pathway. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.Nucleic Acids Research 04/2015; DOI:10.1093/nar/gkv371 · 8.81 Impact Factor
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ABSTRACT: Ig class switching requires cell proliferation and is division linked, but the detailed mechanism is unknown. By analyzing the first switching cells early in the kinetics, our analysis suggested that proliferating B cells had a very short G1 phase (<3.5 h), a total cell cycle time of ∼11 h, and that Ig class switching preferentially occurred in the late G1 or early S phase. Inhibition of cyclin-dependent kinases (CDKs) caused dramatic reduction of switching rate within 6 h. This was associated with less targeting of activation-induced cytidine deaminase (AID) to the Igh locus. Interestingly, ectopically expressed nuclear AID in HeLa cells was preferentially found in the early S phase. Furthermore, in CDK2 hypomorphic cells there was reduced nuclear AID accumulation. Thus, our data are compatible with the idea that division-linked Ig class switching is in part due to CDK2-regulated AID nuclear access at the G1/S border. Copyright © 2015 by The American Association of Immunologists, Inc.The Journal of Immunology 03/2015; DOI:10.4049/jimmunol.1402146 · 5.36 Impact Factor
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ABSTRACT: Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterise a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally-controlled DNA damage response (DDR) characterised by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DNA damage response protein kinases as well as BH3 mimetics promote apoptosis synergistically with Taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.Open Biology 02/2015; 5(3). DOI:10.1098/rsob.140156 · 4.56 Impact Factor