The long-term goal of this proposal is to understand ATM and ATR kinase signaling at DNA replication forks. While ATM kinase is activated by DNA double strand breaks, ATR kinase is induced by single-stranded (ssDNA) gaps. However, crosstalk exists between the pathways and ATM and ATR phosphorylate an overlapping set of substrates. To identify indispensable ATM kinase signaling we used the ATM kinase inhibitors KU55933 and KU60019 to transiently inhibit ATM kinase activity in cells. Using this innovative approach we showed that the consequences of acute ATM kinase inhibition and ATM protein disruption are distinct. Here we show that acute ATM kinase inhibition arrests DNA synthesis. This is surprising since irradiated cells that express no ATM protein do not arrest DNA synthesis due to a defect in the inhibition of late origin firing. The contribution of chain elongation arrest to the intra-S-phase checkpoint has been difficult to establish since lesions that induce the checkpoint also directly arrest replication forks. While recent evidence indicates that ATR signaling can arrest chain elongation, the role of ATM has not been addressed. We hypothesize that ATM kinase activity promotes the steady progression of replication forks and attenuates ATR kinase activity. We propose that acute ATM kinase inhibition impedes the repair of damaged replication forks causing an accumulation of ssDNA gaps that induce ATR kinase signaling and the intra-S-phase checkpoint. This challenges the paradigm that ATM kinase disruption disables the intra-S-phase checkpoint. Here we will combine the use of KU55933 and KU60019 as sharp tools to inhibit ATM kinase signaling with single DNA fiber-based technology that allows the visualization of multiple origins and individual replication forks emerging from those origins. We will undertake the first investigation of origin density and replication fork velocity in cells following acute ATM kinase inhibition and ATM protein disruption.

Public Health Relevance

ATM kinase inhibitors are being developed as clinical radiosensitizing agents. We have shown that ATM kinase has significant roles during the DNA replication process. We propose that the great promise of ATM kinase inhibitors lies in their potential as stand alone agents for the effective treatment of 10,000s of cancers that experience replication stress as a consequence of acquired mutations every year.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Cancer Etiology Study Section (CE)
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Pelroy, Richard
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University of Pittsburgh
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Kiesel, Brian F; Shogan, Jeffrey C; Rachid, Madani et al. (2017) LC-MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR inhibitor AZD6738 in mouse plasma. J Pharm Biomed Anal 138:158-165
Moiseeva, Tatiana N; Gamper, Armin M; Hood, Brian L et al. (2016) Human DNA polymerase ? is phosphorylated at serine-1940 after DNA damage and interacts with the iron-sulfur complex chaperones CIAO1 and MMS19. DNA Repair (Amst) 43:9-17
Villaruz, Liza C; Jones, Helen; Dacic, Sanja et al. (2016) ATM protein is deficient in over 40% of lung adenocarcinomas. Oncotarget 7:57714-57725
Bakkenist, Christopher J; Kastan, Michael B (2015) Chromatin perturbations during the DNA damage response in higher eukaryotes. DNA Repair (Amst) 36:8-12
Bakkenist, Christopher J; Czambel, R Kenneth; Hershberger, Pamela A et al. (2015) A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells. Oncoscience 2:542-54
Bakkenist, Christopher J; Beumer, Jan H; Schmitz, John C (2015) ATM serine-1981 phosphorylation is a plausible biomarker. Cell Cycle 14:3207-8
Vendetti, Frank P; Lau, Alan; Schamus, Sandra et al. (2015) The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo. Oncotarget 6:44289-305
Beumer, Jan H; Fu, Katherine Y; Anyang, Bean N et al. (2015) Functional analyses of ATM, ATR and Fanconi anemia proteins in lung carcinoma : ATM, ATR and FA in lung carcinoma. BMC Cancer 15:649
Teng, Pang-ning; Bateman, Nicholas W; Darcy, Kathleen M et al. (2015) Pharmacologic inhibition of ATR and ATM offers clinically important distinctions to enhancing platinum or radiation response in ovarian, endometrial, and cervical cancer cells. Gynecol Oncol 136:554-61
Forero, Adriana; Giacobbi, Nicholas S; McCormick, Kevin D et al. (2014) Simian virus 40 large T antigen induces IFN-stimulated genes through ATR kinase. J Immunol 192:5933-42

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