Considerable evidence indicates that RB1 loss favourably affects the response of tumours to radiotherapy. Several clinical studies report that absence of RB1 expression predicts treatment success of therapies involving IR, as indicated by prolonged disease-free survival and absence of distant metastasis [33,34,35,36]. RB1 mediates the proliferation block induced by a range of DNA damaging agents and cells with RB1 loss show accelerated death following DNA damage [29,37], suggesting that inhibition of radiation-mediated RB1 activation could be a strategy for radio- sensitization of RB1 positive cancers. The current knowledge as to the signalling that instigates RB1 activation is incomplete and controversial [30,38,39,40]. Here we describe results from a kinome-spanning cell-based screen aimed at the unbiased identification of signalling required for RB1 activation by IR. We identify a group of kinases, hitherto largely unrecognized for their involvement in this context. We character- ize the mode by which they interact with the cellular IR response and document their involvement in facilitating G1-arrest and survival of IR-exposed cells. Results Identification of signalling required for IR–driven RB1 activation To build a screening assay for signalling involved in radiation- mediated RB1 activation we determined the circumstances under which changes in RB1 phosphorylation arise in irradiated cells. We used HCT116 colon-derived carcinoma cells which represent a clinically relevant cancer type for radiation treatment and which express wt RB1. A robust loss of RB1 phosphorylation is seen in these cells between 16 and 24 hours after IR exposure, as indicated by a reduced signal with a phosphorylation-selective antibody for RB1 P-Ser608 (Figure 1A, signal quantification in Figure S1A, A9). This response arose in HCT116 p53+/+ cells but not in isogenic HCT116 p532/2 cells (Figure 1B) for signal quantification see (Figure S1), indicating a critical contribution of TP53 signalling to radiation-mediated RB1 activation. Loss of phosphorylation was seen on other sites, including Ser780 and Ser795 (Figure 1C) and RB1 accumulating in irradiated HCT116 cells had an increased propensity for binding to E2F (Figure S1), indicating general loss of RB1 phosphorylation, and functional activation in these cells. Using the conditions established we developed a 96-well screening assay (Figure 1D) based on quantitative immunofluorescent detection of phosphorylated RB1 in fixed cells. The assay involved use of an antibody selective for Ser780-phosphorylated (PS780) RB1, which proved technically superior to antibodies for other sites in pilot experiments, and determined the percentage of cells with significantly lower than average signal intensity (POS-LoRBPS780) using high- content single cell-based analysis (see Materials and Methods and Figure S4). Transfection of HCT116 cells with siRNA pools targeting TP53 or its downstream target CDKN1A/p21CIP1/WAF1 reduced POS-LoRBPS780 values in these cells more than 6-fold, compared to cells transfected with either a non-targeting siRNA (NT) or without oligonucleotide addition (Mock) (Figure 1E), demonstrating the capacity of the assay to report checkpoint loss following siRNA-based knockdown of relevant signalling components. To uncover unknown signalling required for IR-mediated RB1 activation we screened an siRNA collection targeting all human kinases and additional accessory molecules (779 targets) involved in phospho-proteome regulation (see Table S1 for list of targets). Based on mean POS-LoRBS780 values derived from triplicate runs 59 of the 779 targets reached z scores ,21.5 with 22 targets scoring ,22 (Figure 1F). To identify hits with substantial impact we further graded targets scoring with z,21.5 on the magnitude by Mechanism of G1 Radiation Checkpoint Activation which their respective siRNA pools prevented RB1-PS780 loss. ‘Strong’ targets reduced the mean POS-LoRBPS780 by 2-fold or greater, ‘average’ hits led to a reduction of 2- to 1.6-fold and ‘weak’ hits reduced the average POS-LoRBPS780 between 1.6- and 1.4- fold (see Table S1). In total this yielded 41 hits, with 12 scoring ‘strong’, 18 ‘average’ and 11 ‘weak’. In a screen run in parallel using unirradiated cells none of these hits reached z-scores less than 21.3 and the vast majority scored greater 21 (Figure S1), indicating that the observed radiation-resistant RB1 phosphorylation is not due to target knockdown increasing RB1 phosphorylation in unchallenged cells. Gene names, identifiers and screen data for these hits are listed in the Table S1, alongside data for all targets screened. Gene ontology and pathway association of hits Amongst the hits identified was the TP53 target gene CDKN1A/ p21CIP1/WAF1 [41], predicted from our initial assessment as being critical for RB1 activation, confirming screen performance. The Ataxia telangiectasia mutated (ATM) double stand break (DSB)- activated protein family kinases ATM and ATR, and the checkpoint kinase family kinases CHK1 or CHK2, known to activate TP53 signalling as part of the canonical double stand DNA damage response, did not score, even though these targets were represented in the gene set screened, suggesting that this signalling plays no role in eliciting activation of the checkpoint under investigation. To address if this signalling is indeed unnecessary for RB1 activation following IR we used pharmacological inhibitors for this signalling space (Figure S2). Neither treatment with KU-5593, a selective inhibitor of ATM/ATR, nor the CHK1 selective inhibitor SAR020106 abolished the radiation induced loss of RB1 phosphor- ylation, (Figure S2). As previously observed (Figure 1), radio-resistant RB1 phosphorylation was seen in parallel samples where cells were transfected with siRNA targeting TP53. Both Ku-5593 and SAR020106 inhibited autocatalytic activity of CHK1 (Figure S2), indicative that they were effective in blocking damage-driven signal transduction in the cell line and at the dose used. Analysis of lysates from cells treated with these inhibitors provided corroborating evidence, revealing net loss of RB1 phosphorylation following IR exposure comparable to that of Mock-treated cells. Together these results corroborate the critical requirement of TP53 and p21CIP1/ WAF1 as the signal executing axis yet indicate that signalling distinct from the canonical TP53 activating DSB signalling is involved in controlling radiation-mediated RB1 checkpoint activation. To obtain information as to the type of signalling that was detected in the screen we probed for the association of the identified hits with known signalling pathway ontology. To do so we searched for representation of hits within defined pathways and processes using the NIH Database for Annotation, Visualization and Integrated Discovery (DAVID), http://david.abcc.ncifcrf. gov/home.jsp. This revealed considerable representation within MAPK and calcium signalling (Figure 2A) along with membrane receptor signalling ontology in which both MAPK and calcium signalling play a role. Overall, 23 of the 41 hits (57%) were accounted for by these pathway categories. 18 hits (43%) were not represented within the analysis output, indicating that the screen also identified components that do not significantly cluster within the pathways and processes considered in the database interro- gated. A number of pathways although strongly represented in the screened gene set were not reflected in the hit list, indicating selectivity of the screen (Figure 2B). To confirm validity of the hits and ensuing predictions, we sought hit confirmation using individual siRNA duplexes. We confined this analysis to hits that had scored as either strong or average in the primary screen, regardless of whether they were associated within defined pathway ontology or not. PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31627