Molecular and Cellular Pathobiology Loss of PPP 2 R 2 A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition

Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G1 to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non–small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic. Cancer Res; 72(24); 1–11. 2012 AACR. Introduction Genome stability is essential for the prevention of undue cellular death and cancer development. To maintain genome integrity, cells have evolved multiple DNA repair pathways (1). One of themost powerful activators of theDNA repair response is double-strand breaks (DSB). Initiation of DSB repair is controlled by the phosphoinositide 3-kinase–like kinase (PIKK) family. A wave of phosphorylation events radiating from PIKKs is amplified to convey the signals to a large number of substrates. Although this cascade has been studied in a great detail, the biologic relevance of many of these phosphorylation events and the mechanisms that control their downregulation remain unknown (2). It is conceivable that Ser/Thr protein phosphatases could be responsible for keeping proteins involved in DNA repair response in an inactive state under normal conditions or for inactivating the signaling once DNA has been repaired. However, phosphorylation of a number of PIKKs, including ataxia telangiectasia mutated (ATM), ATR, and CHK2, oscillates during the DNA repair process (3, 4), suggesting that phosphatases may serve not only as negative regulators but also as active modulators of DNA damage response. Protein phosphatase 2A (PP2A) refers to a large family of heterotrimeric Ser/Thr phosphatases. The PP2A core enzyme consists of a catalytic C subunit and a structural A subunit. In mammals, 2 distinct genes encode closely related versions of both the PP2A A (Aa/PPP2R1A and Ab/PPP2R1B) and C (Ca/ PPP2CA and Cb/PPP2CB) subunits. The AC dimer recruits a third regulatory B subunit, which is responsible for the substrate specificity and function of the PP2A heterotrimeric complex. Four unrelated families of B subunits have been identified to date: B/B55/PPP2R2A, B0/B56/PR61/PPP2R5, B00/PR72/PPP2R3, and Striatin/STRN. Approximately 100 distinct complexes can be formed through combinatorial association of these subunits, and it is believed that specific PP2A complexes mediate particular physiologic functions (5, 6). PP2A has been directly implicated in the negative regulation of DSB DNA repair proteins, including g-H2AX, ATM, CHK1, and CHK2 (2). However, consistent with the idea that protein phosphatases are not just negative regulators of DNA repair signaling, selective inhibition of PP2A activity impairs DNA repair (7–9). PP2A function is also essential for activation of cell-cycle checkpoints in response to irradiation (10, 11). One potential explanation for this apparent discrepancy is that several distinct PP2A complexes may modulate different steps of the DNA repair process. Here, we assessed the role of specific PP2A complexes in control of DSB repair and identify PPP2R2A as a critical effector of the homologous recombination (HR) repair through modulation of ATM phosphorylation. Critically, defects of HR DNA repair in PPP2R2A-depleted cells dramatically increased Authors' Affiliations: VIB Center for the Biology of Disease; Center for Human Genetics, KU Leuven, Leuven, Belgium; and Department of Toxicology, Faculty of Preventive Medicine, Guangdong Provincial Key Laboratory of Food, Nutrition andHealth, School of Public Health, Sun Yatsen University, Guangzhou, China Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). CorrespondingAuthor:AnnaSablina, CMEDepartment, KULeuven,O&N I Herestraat 49, bus 602, Leuven, Belgium 3000. Phone: 32-163-30790; Fax: 32-163-30145; E-mail: Anna.Sablina@cme.vib-kuleuven.be doi: 10.1158/0008-5472.CAN-12-1667 2012 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on January 28, 2018. © 2012 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667