Mechanism-Based Screen Establishes Signalling Framework for DNA Damage-Associated G1 Checkpoint Response Elizabeth Richardson1,2, Simon R. Stockwell1,2, He Li2, Wynne Aherne3, Maria Emanuela Cuomo1,2, Sibylle Mittnacht1,2* 1 Department of Cancer Biology, UCL Cancer Institute, London, United Kingdom, 2 Division of Cancer Biology, The Institute of Cancer Research, London, United Kingdom, 3 Division of Cancer Therapeutics, The Institute of Cancer Research, London, United Kingdom Abstract DNA damage activates checkpoint controls which block progression of cells through the division cycle. Several different checkpoints exist that control transit at different positions in the cell cycle. A role for checkpoint activation in providing resistance of cells to genotoxic anticancer therapy, including chemotherapy and ionizing radiation, is widely recognized. Although the core molecular functions that execute different damage activated checkpoints are known, the signals that control checkpoint activation are far from understood. We used a kinome-spanning RNA interference screen to delineate signalling required for radiation-mediated retinoblastoma protein activation, the recognized executor of G1 checkpoint control. Our results corroborate the involvement of the p53 tumour suppressor (TP53) and its downstream targets p21CIP1/WAF1 but infer lack of involvement of canonical double strand break (DSB) recognition known for its role in activating TP53 in damaged cells. Instead our results predict signalling involving the known TP53 phosphorylating kinase PRPK/TP53RK and the JNK/p38MAPK activating kinase STK4/MST1, both hitherto unrecognised for their contribution to DNA damage G1 checkpoint signalling. Our results further predict a network topology whereby induction of p21CIP1/WAF1 is required but not sufficient to elicit checkpoint activation. Our experiments document a role of the kinases identified in radiation protection proposing their pharmacological inhibition as a potential strategy to increase radiation sensitivity in proliferating cancer cells. Citation: Richardson E, Stockwell SR, Li H, Aherne W, Cuomo ME, et al. (2012) Mechanism-Based Screen Establishes Signalling Framework for DNA Damage- Associated G1 Checkpoint Response. PLoS ONE 7(2): e31627. doi:10.1371/journal.pone.0031627 Editor: Sue Cotterill, St. Georges University of London, United Kingdom Received May 24, 2011; Accepted January 16, 2012; Published February 27, 2012 Copyright: 2012 Richardson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported through Cancer Research UK grant numbers CRC278X and CRM048. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sibylle.mittnacht@cancer.ucl.ac.uk Introduction DNA damage through exposure to ionising radiation (IR) is an important tool in cancer therapy. Radiotherapy features in the treatment of greater than 50% of all cancers and IR is considered the most effective treatment option for inoperable solid tumours [1,2]. Although objective responses are frequent, long-term remission is not often seen, and patients commonly relapse with tumour re-growth following cessation of treatment [3]. Increasing evidence suggests that the genetic makeup of tumours critically influence the IR-sensitivity of cancer tissue and the duration of remission in therapies involving IR [4]. Loss of either damage repair [5] or damage-inducible cell cycle checkpoint control [6] enhances IR sensitivity, suggesting that both repair efficacy and checkpoint activation confer radioprotection. Other evidence indicates that preferential activation of checkpoint control provides resistance to cancer stem cells [7]. Hence inhibition of repair or checkpoint signalling has been proposed as a strategy for enhancing the response of cancers to radiotherapy [8,9]. DNA damage-inducible cell cycle checkpoints transiently delay cell cycle progression in proliferating cells, presumably providing time for repair [10,11]. DNA damage checkpoint control arises at multiple points of the cell cycle including late G1 (G1), intra S phase, and the G2 phase [12]. Recent years have seen considerable progress in elucidating signalling involved in the different types of checkpoint control. Checkpoint kinases 1 and 2 (CHK1/2) are key executors involved in delaying S and G2/M transit [13,14,15,16]. CHKs phosphorylate, and thus inhibit, the dual specificity phosphatases CDC25B and A [17,18,19,20] required for activa- tion of the CDK2 and CDK1 cyclin-dependent kinases which drive DNA synthesis and entry of cells into M phase respectively. Other work demonstrates involvement of MAPKAP-kinase2 (MK2) and MK2-dependent GADD45A biosynthesis [21,22], and a role for the p53 tumour suppressor protein TP53 in the maintenance of the G2 checkpoint response [23,24]. G1 checkpoint activation is thought to involve the retinoblas- toma tumour-suppressor (RB1) and its paralogues. RB1 inhibits the transcription of gene products required for S phase entry, amongst them the CDK2 activating cyclins E and A [25], and it stabilizes the CDK inhibitory proteins p27KIP1/CDKN1B and p21CIP1/WAF1/CDKN1A [26]. Exposure of cells to IR leads to accumulation of RB1 in its active, underphosphorylated form [27,28]. G1 checkpoint activation in irradiated cells is likely to be of dual significance. In response to DNA damage, G1 checkpoint execution may delay progression of G1 cells from entering S phase [29,30]. G1 checkpoint activation also underlies ‘‘adaptation’’, which follows escape of damaged cells from G2 arrest [31,32]. PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31627