Delineating the DNA damage response using systems biology approaches

Cellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer formation and ageing as well as genotoxic stress-induced cancer therapy. To do justice to the multifaceted cellular changes, elicited by DNA damage, use of high-throughput techniques and integration with bioinformatics tools is of great value. This thesis summarizes recent advances in the field of systems biology studies of the DNA damage response and furthermore shows integrated approaches of the study of DNA damage response signaling networks in embryonic stem and cancer cells. By integration of transcriptional changes and the phosphorylation and metabolic response of cisplatin-treated embryonic stem cells, with RNAi-based knockdown screens we identify novel DNA damage response signaling networks, linking process such as Wnt signaling, translation arrest or altered metabolic pathways to the cellular response to DNA damage. Furthermore, genes, whose knockdown sensitizes embryonic stem cells to DNA damage-induced killing, are tested in cancer cells of varying genetic backgrounds identifying a small subset of genes, which represent potential drug targets for sensitization of cancer cells. Altogether, our systems approach for studying the DNA damage response identifies novel DNA damage-induced signaling networks and molecules, which modulate survival in the presence of DNA damage, potentially providing new targets for therapeutic intervention or biomarker discovery.Research was supported by the Netherlands Genomics Initiative /Netherlands Organization for Scientific Research (NWO): nr 050-060-510. Support for thesis printing came from Havelland Stiftung, Berlin.UBL - phd migration 201

Annex 1 - Glossary

This glossary defines some specific terms as the Lead Authors intend them to be interpreted in the context of this report

2°C and SDGs: United they stand, divided they fall?

The adoption of the Sustainable Development Goals (SDGs) and the new international climate treaty could put 2015 into the history books as a defining year for setting human development on a more sustainable pathway. The global climate policy and SDG agendas are highly interconnected: the way that the climate problem is addressed strongly affects the prospects of meeting numerous other SDGs and vice versa. Drawing on existing scenario results from a recent energy-economy-climate model inter-comparison project, this letter analyses these synergies and (risk) trade-offs of alternative 2 °C pathways across indicators relevant for energy-related SDGs and sustainable energy objectives. We find that limiting the availability of key mitigation technologies yields some co-benefits and decreases risks specific to these technologies but greatly increases many others. Fewer synergies and substantial trade-offs across SDGs are locked into the system for weak short-term climate policies that are broadly in line with current Intended Nationally Determined Contributions (INDCs), particularly when combined with constraints on technologies. Lowering energy demand growth is key to managing these trade-offs and creating synergies across multiple energy-related SD dimensions. We argue that SD considerations are central for choosing socially acceptable 2 °C pathways: the prospects of meeting other SDGs need not dwindle and can even be enhanced for some goals if appropriate climate policy choices are made. Progress on the climate policy and SDG agendas should therefore be tracked within a unified framework

Identification of Cisplatin-Regulated Metabolic Pathways in Pluripotent Stem Cells

The chemotherapeutic compound, cisplatin causes various kinds of DNA lesions but also triggers other pertubations, such as ER and oxidative stress. We and others have shown that treatment of pluripotent stem cells with cisplatin causes a plethora of transcriptional and post-translational alterations that, to a major extent, point to DNA damage response (DDR) signaling. The orchestrated DDR signaling network is important to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of the DDR in stem cells contributes to ageing and cancer. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES) cells treated for different time periods with cisplatin. We then integrated metabolomics with transcriptomics analyses and connected cisplatin-regulated metabolites with regulated metabolic enzymes to identify enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism. Silencing of identified proline metabolic and catabolic enzymes indicated that altered proline metabolism serves as an adaptive, rather than a toxic response. A group of enriched metabolic pathways clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. Enzymes and metabolites with pro- or anti-oxidant functions were also enriched but enhanced levels of reactive oxygen species were not measured in cisplatin-treated ES cells. Lastly, a number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that are responsive to cisplatin and may serve as signaling modules in the DDR in pluripotent stem cells

Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response

2 °C and SDGs: united they stand, divided they fall?

The adoption of the Sustainable Development Goals (SDGs) and the new international climate treaty could put 2015 into the history books as a defining year for setting human development on a more sustainable pathway. The global climate policy and SDG agendas are highly interconnected: the way that the climate problem is addressed strongly affects the prospects of meeting numerous other SDGs and vice versa. Drawing on existing scenario results from a recent energy-economy-climate model inter-comparison project, this letter analyses these synergies and (risk) trade-offs of alternative 2 °C pathways across indicators relevant for energy-related SDGs and sustainable energy objectives. We find that limiting the availability of key mitigation technologies yields some co-benefits and decreases risks specific to these technologies but greatly increases many others. Fewer synergies and substantial trade-offs across SDGs are locked into the system for weak short-term climate policies that are broadly in line with current Intended Nationally Determined Contributions (INDCs), particularly when combined with constraints on technologies. Lowering energy demand growth is key to managing these trade-offs and creating synergies across multiple energy-related SD dimensions. We argue that SD considerations are central for choosing socially acceptable 2 °C pathways: the prospects of meeting other SDGs need not dwindle and can even be enhanced for some goals if appropriate climate policy choices are made. Progress on the climate policy and SDG agendas should therefore be tracked within a unified framework.EC/FP7/265139/EU/Assessment of Climate Change Mitigation Pathways and Evaluation of the Robustness of Mitigation Cost Estimates/AMPEREEC/H2020/642147/EU/Linking Climate and Development Policies - Leveraging International Networks and Knowledge Sharing/CD-LINK