There is a need for alternative methods to replace, reduce and refine (3R) animal experimentation. Combining experimental data from high-throughput in vitro studies with in silico modeling is a... Show moreThere is a need for alternative methods to replace, reduce and refine (3R) animal experimentation. Combining experimental data from high-throughput in vitro studies with in silico modeling is a promising approach to unravel the effect of chemicals on living cells and to gain a better understanding of the processes leading to adverse effects. Exposure to chemicals can activate various stress response pathways that limit the amount of cellular damage, help cells to recover or orchestrate irreversible cell fates such as apoptosis. In this thesis, we use experimental data and current knowledge on stress pathway activation and cell fate to create different types of computational models. With these models, we mathematically describe intracellular protein signaling cascades activated upon exposure to various compounds and their link to cell fate. In this way, we integrate molecular-level biological processes to cell-level phenomena such as cell cycle progression, senescence and necrosis, and generate new hypotheses about the mechanisms underlying adversity. Show less
Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of... Show moreAnthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here, we show that the extensively used anthracyclines Doxorubicin, Daunorubicin, and Epirubicin decrease the transcription of nuclear factor kappa B (NF-kappa B)-dependent gene targets, but not interferon-responsive genes in primary mouse (Mus musculus) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-kappa B subunit RelA and its DNA-binding sites. The anthracycline variants Aclarubicin, Doxorubicinone, and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis. Show less
Cancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low ... Show moreCancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low (LD; 0.1 Gy) or a high (HD; 1 Gy) dose of X-rays, we show that, although the DNA damage response (DDR) displayed dose proportionality, many other molecular and cellular responses did not. Phosphoproteomics uncovered a novel mode of phospho-signaling via S12-PPP1R7, and large-scale dephosphorylation events that regulate mitotic exit control in undamaged cells and the G2/M checkpoint upon IR in a dose-dependent manner. The phosphoproteomics of irradiated DNA double-strand breaks (DSBs) repair-deficient cells unveiled extended phospho-signaling duration in either a dose-dependent (DDR signaling) or independent (mTOR-ERK-MAPK signaling) manner without affecting signal magnitude. Nascent transcriptomics revealed the transcriptional activation of genes involved in NRF2-regulated antioxidant defense, redox-sensitive ERK-MAPK signaling, glycolysis and mitochondrial function after LD, suggesting a prominent role for reactive oxygen species (ROS) in molecular and cellular responses to LD exposure, whereas DDR genes were prominently activated after HD. However, how and to what extent the observed dose-dependent differences in molecular and cellular responses may impact cancer development remain unclear, as the induction of chromosomal damage was found to be dose-proportional (10-200 mGy). Show less
All cellular organisms contain genomic DNA which provides the instructions for their correct development and functioning. Damage to this DNA may interfere with critical cellular processes such as... Show moreAll cellular organisms contain genomic DNA which provides the instructions for their correct development and functioning. Damage to this DNA may interfere with critical cellular processes such as transcription and replication and has the potential to drive mutagenesis. In turn, this may underlie inherited disorders and accelerate progression of diseases such as cancer and neurodegenerative disorders. The protection of cells and organisms against these devastating effects of DNA damage relies on the DNA damage response (DDR), which comprises a complex network of signaling and repair pathways that coordinate the sensing, signaling and repair of DNA lesions while accommodating suitable adjustments in for instance chromatin structure and cell cycle progression. Not only does the DDR dictate the appropriate repair pathway for several types of DNA damage, including DNA double-strand breaks (DSB), it also modulates replication fork surveillance mechanisms in response to DNA replication stress (RS). While many core proteins have been studied in detail, the full repertoire of factors involved in these pathways remains unknown. Clearly, extending our knowledge on regulators of the DDR will contribute to our understanding of the development, and possibly the treatment, of the numerous disorders that are associated with defects in the DDR. The research described in this thesis has successfully identified and characterized novel factors in DSB repair and the RS response. Show less
Sampadi, B.; Mullenders, L.H.F.; Vrieling, H. 2022
Background: Although cancer risk is assumed to be linear with ionizing radiation (IR) dose, it is unclear to what extent low doses (LD) of IR from medical and occupational exposures pose a cancer... Show moreBackground: Although cancer risk is assumed to be linear with ionizing radiation (IR) dose, it is unclear to what extent low doses (LD) of IR from medical and occupational exposures pose a cancer risk for humans. Improved mechanistic understanding of the signaling responses to LD may help to clarify this uncertainty. Here, we per -formed quantitative mass spectrometry-based proteomics and phosphoproteomics experiments, using mouse embryonic stem cells, at 0.5 h and 4 h after exposure to LD (0.1 Gy) and high doses (HD; 1 Gy) of IR. Results: The proteome remained relatively stable (29; 0.5% proteins responded), whereas the phosphoproteome changed dynamically (819; 7% phosphosites changed) upon irradiation. Dose-dependent alterations of 25 IR-responsive proteins were identified, with only four in common between LD and HD. Mitochondrial metabolic proteins and pathways responded to LD, whereas transporter proteins and mitochondrial uncoupling pathways responded to HD. Congruently, mitochondrial respiration increased after LD exposure but decreased after HD exposure. While the bulk of the phosphoproteome response to LD (76%) occurred already at 0.5 h, an equivalent proportion of the phosphosites responded to HD at both time points. Motif, kinome/phosphatome, kinase-substrate, and pathway analyses revealed a robust DNA damage response (DDR) activation after HD exposure but not after LD exposure. Instead, LD-irradiation induced (de)phosphorylation of kinases, kinase-substrates and phosphatases that predominantly respond to reactive oxygen species (ROS) production. Conclusion: Our analyses identify discrete global proteome and phosphoproteome responses after LD and HD, uncovering novel proteins and protein (de)phosphorylation events involved in the dose-dependent ionizing ra-diation responses. Show less
Sampadi, B.; Mullenders, L.H.F.; Vrieling, H. 2021
The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling... Show moreThe influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods-strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology. Show less
Genotoxic stressors may induce various types of DNA damage triggering the activation of the DNA damage response. However, the dose-dependency and the drug compound-specificity of the activated... Show moreGenotoxic stressors may induce various types of DNA damage triggering the activation of the DNA damage response. However, the dose-dependency and the drug compound-specificity of the activated responses are unclear. Here, I first investigated the phospho-signalling responses induced by four chemical stressors with distinct modes of action and uncovered that, at equitoxic doses, even two ‘similar’ DNA damaging agents induced discrete and complex phosphorylation signalling cascades. Next, I investigated the ionising radiation (IR) dose-dependent responses using multi-omics and systems analyses to investigate the molecular and cellular responses to low and high doses of IR. I uncovered that the IR induced molecular responses have different dose-effect relationships. A minor part of the phosphorylation signalling and transcription involved in the DSB-related responses displayed a linear dose-effect relationship. Contrastingly, the reactive oxygen species (ROS) production-related molecular and cellular responses and DNA replication stress revealed complex dose-response relationships. LD uniquely activates ROS-related responses at multiple molecular levels, including proteome, phosphoproteome, nascent transcriptome and metabolome. Further work will be required to determine how these differences in molecular and cellular responses observed over hours after low and high doses impact the risk for cancer development that occurs over months and years. Show less
DNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double-strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent... Show moreDNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double-strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent recruitment of the deubiquitylating enzyme ataxin-3 to DSBs, unlike recruitment of the ubiquitin ligase RNF4, additionally depends on poly [ADP-ribose] polymerase 1 (PARP1)-mediated poly(ADP-ribosyl)ation (PARylation). The co-dependence of ataxin-3 recruitment on PARylation and SUMOylation temporally confines ataxin-3 to DSBs immediately after occurrence of DNA damage. We propose that this mechanism ensures that ataxin-3 prevents the premature removal of DNA repair proteins only during the early phase of the DSB response and does not interfere with the subsequent timely displacement of DNA repair proteins by RNF4. Thus, our data show that PARylation differentially regulates SUMO-dependent recruitment of ataxin-3 and RNF4 to DSBs, explaining how both proteins can play a stimulatory role at DSBs despite their opposing activities. Show less
Leeksma, A.C.; Derks, I.A.M.; Kasem, M.H.; Kilic, E.; Klein, A. de; Jager, M.J.; ... ; Eldering, E. 2021
Recurrent mutations in splicing factor 3B subunit 1 (SF3B1) have been identified in several malignancies and are associated with an increased expression of 3' cryptic transcripts as a result of... Show moreRecurrent mutations in splicing factor 3B subunit 1 (SF3B1) have been identified in several malignancies and are associated with an increased expression of 3' cryptic transcripts as a result of alternative branchpoint recognition. A large fraction of cryptic transcripts associated with SF3B1 mutations is expected to be sensitive for RNA degradation via nonsense-mediated mRNA decay (NMD). Several studies indicated alterations in various signaling pathways in SF3B1-mutated cells, including an impaired DNA damage response (DDR) in chronic lymphocytic leukemia (CLL). In this study, we investigated isogenic cell lines and treatment naive primary CLL samples without any TP53 and/or ATM defect, and found no significant effects of SF3B1 mutations on the ATM/p53 response, phosphorylation of H2AX and sensitivity to fludarabine. Cryptic transcripts associated with SF3B1 mutation status were observed at relatively low levels compared to the canonical transcripts and were validated as target for mRNA degradation via NMD. Expression of cryptic transcripts increased after NMD inhibition. In conclusion, our results confirm involvement of NMD in the biological effects of SF3B1 mutations. Further studies may elucidate whether SF3B1-mutant patients could benefit from NMD modulatory agents. Show less
Our cells are continuously challenged by numerous external as well as internal hazards that, if not dealt with in an appropriate manner, may interfere with critical processes and underlie disease.... Show moreOur cells are continuously challenged by numerous external as well as internal hazards that, if not dealt with in an appropriate manner, may interfere with critical processes and underlie disease. Therefore, they heavily rely on protective mechanisms that recognize and counteract potential risks. For example, the immune system provides a first line of defense against pathogens, which upon host invasion could cause serious illness. In addition, the well-coordinated networks of the DNA damage response detect and remove damaged nucleotides that could drive mutagenesis and thereby provoke inherited disorders, cancer or ageing-related diseases. The correct activation, execution and completion of the implicated pathways, as wel as their crosstalk, is of key importance and necessitates the well-timed and -positioned presence of proteins with the desired functionality. To a great extent, this is established by post-translational modifications (PTMs) that fine-tune protein functionality. We used a combination of biological, biochemical and microscopic approaches to determine the contribution of several PTMs to the regulation of the mechanisms that safeguard our cells. Show less
Small ubiquitin-like modifiers (SUMOs) are small proteins that can be covalently attached to hundreds of target proteins. This post-translational modification can alter protein binding sites and... Show moreSmall ubiquitin-like modifiers (SUMOs) are small proteins that can be covalently attached to hundreds of target proteins. This post-translational modification can alter protein binding sites and therefore influence function, localization or stability of the target protein. This thesis describes novel roles of SUMOylation in three important cellular processes, proteostasis, centromere integrity and the DNA damage response. SUMOylation promotes proteostasis after heat-shock in cooperation with the HSF-1-regulated chaperone network. Furthermore, we show that the SUMO specific isopeptidase SENP6 is responsible for the deSUMOylation of multiple subunits of the constitutive centromere-associated network (CCAN), an important protein complex that provides the basis for the assembly of the kinetochore during mitosis. Failure of deSUMOylation results in an accumulation of SUMO chains on the CCAN proteins and consequently less efficient localization to the centromere. SUMOylation has multiple previously described roles in the DNA damage response. Here, we demonstrate that the protein Cockayne Syndrome B (CSB) is the highest SUMOylated protein in response to UV-induced DNA damage. SUMOylation promotes efficient localization of CSB to the damage sites and contributes to efficient repair. Show less
Upon the induction of DNA damage, cells initiate a protective response, referred to as the DNA damage response (DDR), to repair DNA damage and maintain genome integrity. This response is driven... Show moreUpon the induction of DNA damage, cells initiate a protective response, referred to as the DNA damage response (DDR), to repair DNA damage and maintain genome integrity. This response is driven and regulated by posttranslational protein modifications and chromatin remodeling events. Mutations or aberrant expression of chromatin modifying proteins not only impacts on the DDR, but also causes human diseases with severe clinical phenotypes, illustrating the importance of these proteins for genome stability maintenance and human health. Largely unclear is, however, which and how chromatin modifying enzymes control the complex DDR pathways and in this manner prevent the onset of disease. To this end, we employed cross-disciplinary approaches that combined cell biological, biochemical and microscopic methods to identify histone modifying enzymes, chromatin remodelers as well as other DDR proteins and elucidate their mechanistic role in the response to DNA doublestrand breaks (DSBs) and disease prevention. Show less
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... Show moreCellular 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. Show less
