Fanconi anaemia (FA) is a rare chromosomal-instability syndrome caused by mutations of any of the 22 known FA DNA-repair genes. FA individuals have an increased risk of head-and-neck squamous-cell-carcinomas (HNSCC), often fatal. Systemic intolerance to standard cisplatin-based protocols due to somatic-cell hypersensitivity underscores the urgent need to develop novel therapies. Here, we performed unbiased siRNA screens to unveil genetic interactions synthetic-lethal with FA-pathway deficiency in FA-patient HNSCC cell lines. We identified based on differential-lethality scores between FA-deficient and FA-proficient cells, next to common-essential genes such as PSMC1, PSMB2, and LAMTOR2, the otherwise non-essential RBBP9 gene. Accordingly, low dose of the FDA-approved RBBP9-targeting drug Emetine kills FA-HNSCC. Importantly both RBBP9-silencing as well as Emetine spared non-tumour FA cells. This study provides a minable genome-wide analyses of vulnerabilities to address treatment challenges in FA-HNSCC. Our investigation divulges a DNA-cross-link-repair independent lead, RBBP9, for targeted treatment of FA-HNSCCs without systemic toxicity.

Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.

The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1-proteins involved in the signaling axis of mammalian DSB repair-in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.

Hyperthermia - application of supra-physiological temperatures to cells, tissues or organs - is a pleiotropic treatment that affects most aspects of cellular metabolism, but its effects on DNA are of special interest in the context of cancer research and treatment. Hyperthermia inhibits repair of various DNA lesions, including double-strand breaks (DSBs), making it a powerful radio- and chemosensitizer, with proven clinical efficacy in therapy of various types of cancer, including tumors of head and neck, bladder, breast and cervix. Among the challenges for hyperthermia-based therapies are the transient character of its effects, the technical difficulties in maintaining uniformly elevated tumor temperature and the acquisition of thermotolerance. Approaches to reduce or eliminate these challenges could simplify the application of hyperthermia, boost its efficacy and improve treatment outcomes. Here we show that a single, short treatment with a relatively low dose of HSP90 inhibitor Ganetespib potentiates cytotoxic as well as radio- and chemosensitizing effects of hyperthermia and reduces thermotolerance in cervix cancer cell lines. Ganetespib alone, applied at this low dose, has virtually no effect on survival of non-heated cells. Our results thus suggest that HSP90 inhibition can be a safe, simple and efficient approach to improving hyperthermia treatment efficacy and reducing thermotolerance, paving the way for in vivo studies.

Homologous recombination (HR) is an essential, widely conserved mechanism that utilizes a template for accurate repair of DNA breaks. Some early HR models, developed over five decades ago, anticipated single-strand breaks (nicks) as initiating lesions. Subsequent studies favored a more double-strand break (DSB)-centered view of HR initiation and at present this pathway is primarily considered to be associated with DSB repair. However, mounting evidence suggests that nicks can indeed initiate HR directly, without first being converted to DSBs. Moreover, recent studies reported on novel branches of nick-initiated HR (nickHR) that rely on single-, rather than double-stranded repair templates and that are characterized by mechanistically and genetically unique properties. The physiological significance of nickHR is not well documented, but its high-fidelity nature and low mutagenic potential are relevant in recently developed, precise gene editing approaches. Here, we review the evidence for stimulation of HR by nicks, as well as the data on the interactions of nickHR with other DNA repair pathways and on its mechanistic properties. We conclude that nickHR is a bona-fide pathway for nick repair, sharing the molecular machinery with the canonical HR but nevertheless characterized by unique properties that secure its inclusion in DNA repair models and warrant future investigations.

Most proteins involved in the DNA double-strand break response (DSBR) accumulate at the damage sites, where they perform functions related to damage signaling, chromatin remodeling and repair. Over the last two decades, studying the accumulation of many DSBR proteins provided information about their functionality and underlying mechanisms of action. However, comparison and systemic interpretation of these data is challenging due to their scattered nature and differing experimental approaches. Here, we extracted, analyzed and compared the available results describing accumulation of 79 DSBR proteins at sites of DNA damage, which can be further explored using Cumulus (http://www.dna-repair.live/cumulus/)-the accompanying interactive online application. Despite large inter-study variability, our analysis revealed that the accumulation of most proteins starts immediately after damage induction, occurs in parallel and peaks within 15-20 min. Various DSBR pathways are characterized by distinct accumulation kinetics with major non-homologous end joining proteins being generally faster than those involved in homologous recombination, and signaling and chromatin remodeling factors accumulating with varying speeds. Our meta-analysis provides, for the first time, comprehensive overview of the temporal organization of the DSBR in mammalian cells and could serve as a reference for future mechanistic studies of this complex process.

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Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

Cis-diamminedichloroplatinum(II) (cisplatin, cDDP) is an effective chemotherapeutic agent that induces DNA double strand breaks (DSBs), primarily in replicating cells. Generally, such DSBs can be repaired by the classical or backup non-homologous end joining (c-NHEJ/b-NHEJ) or homologous recombination (HR). Therefore, inhibiting these pathways in cancer cells should enhance the efficiency of cDDP treatments. Indeed, inhibition of HR by hyperthermia (HT) sensitizes cancer cells to cDDP and in the Netherlands this combination is a standard treatment option for recurrent cervical cancer after previous radiotherapy. Additionally, cDDP has been demonstrated to disrupt c-NHEJ, which likely further increases the treatment efficacy. However, if one of these pathways is blocked, DSB repair functions can be sustained by the Poly-(ADP-ribose)-polymerase1 (PARP1)-dependent b-NHEJ. Therefore, disabling b-NHEJ should, in principle, further inhibit the repair of cDDP-induced DNA lesions and enhance the toxicity of thermochemotherapy. To explore this hypothesis, we treated a panel of cancer cell lines with HT, cDDP and a PARP1-i and measured various end-point relevant in cancer treatment. Our results demonstrate that PARP1-i does not considerably increase the efficacy of HT combined with standard, commonly used cDDP concentrations. However, in the presence of a PARP1-i, ten-fold lower concentration of cDDP can be used to induce similar cytotoxic effects. PARP1 inhibition may thus permit a substantial lowering of cDDP concentrations without diminishing treatment efficacy, potentially reducing systemic side effects.

DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of (nick)HR are largely unexplored. Here, we applied Cas9 nickases to study (nick)HR in mammalian cells. We find that (nick)HR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing.

The currently available arsenal of anticancer modalities includes many DNA damaging agents that can kill malignant cells. However, efficient DNA repair mechanisms protect both healthy and cancer cells against the effects of treatment and contribute to the development of drug resistance. Therefore, anti-cancer treatments based on inflicting DNA damage can benefit from inhibition of DNA repair. Hyperthermia – treatment at elevated temperature – considerably affects DNA repair, among other cellular processes, and can thus sensitize (cancer) cells to DNA damaging agents. This effect has been known and clinically applied for many decades, but how heat inhibits DNA repair and which pathways are targeted has not been fully elucidated. In this review we attempt to summarize the known effects of hyperthermia on DNA repair pathways relevant in clinical treatment of cancer. Furthermore, we outline the relationships between the effects of heat on DNA repair and sensitization of cells to various DNA damaging agents.

Colorectal cancer (CRC) development is in most cases marked by the accumulation of genomic alterations including gain of the entire q‐arm of chromosome 13. This aberration occurs in 40%–60% of all CRC and is associated with progression from adenoma to carcinoma. To date, little is known about the effect of the 13q amplicon on the expression of the therein located genes and their functional relevance. We therefore aimed to identify candidate genes at the 13q amplicon that contribute to colorectal adenoma to carcinoma progression in a gene dosage‐dependent manner. Integrative analysis of whole genome expression and DNA copy number signatures resulted in the identification of 36 genes on 13q of which significant overexpression in carcinomas compared with adenomas was linked to a copy number gain. Five genes showing high levels of overexpression in carcinomas versus adenomas were further tested by quantitative reverse transcription‐PCR in two independent sample sets of colorectal tumors (n = 40 and n = 47). DIS3 and LRCH1 revealed significant overexpression in carcinomas compared with adenomas in a 13q gain dependent manner. Silencing of DIS3 affected important tumorigenic characteristics such as viability, migration, and invasion. In conclusion, significant overexpression of DIS3 and LRCH1 associated with adenoma to carcinoma progression is linked to the CRC specific gain of 13q. The functional relevance of this copy number aberration was corroborated for DIS3, thereby identifying this gene as novel candidate oncogene contributing to the 13q‐driven adenoma to carcinoma progression. © 2014 Wiley Periodicals, Inc.

Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the integrity of the genome. Although SSBs are usually repaired by dedicated pathways, they can also trigger homologous recombination (HR), an error-free pathway generally associated with DSB repair. While HR-mediated DSB repair has been extensively studied, the mechanisms of HR-mediated SSB repair are less clear. This chapter describes a protocol to investigate SSB-induced HR in mammalian cells employing the DR-GFP reporter, which has been widely used in DSB repair studies, together with an adapted bacterial CRISPR/Cas system.

Dendritic cells (DCs) orchestrate antibody-mediated responses to combat extracellular pathogens including parasites by initiating T helper cell differentiation. Here we demonstrate that carbohydrate-specific signalling by DC-SIGN drives follicular T helper cell (TFH) differentiation via IL-27 expression. Fucose, but not mannose, engagement of DC-SIGN results in activation of IKKε, which collaborates with type I IFNR signalling to induce formation and activation of transcription factor ISGF3. Notably, ISGF3 induces expression of IL-27 subunit p28, and subsequent IL-27 secreted by DC-SIGN-primed DCs is pivotal for the induction of Bcl-6(+)CXCR5(+)PD-1(hi)Foxp1(lo) TFH cells, IL-21 secretion by TFH cells and T-cell-dependent IgG production by B cells. Thus, we have identified an essential role for DC-SIGN-induced ISGF3 by fucose-based PAMPs in driving IL-27 and subsequent TFH polarization, which might be harnessed for vaccination design.

One of the hallmarks of cancer is genomic instability controlled by cell cycle checkpoints. The G1 and G2 checkpoints allow DNA damage responses, whereas the mitotic checkpoint enables correct seggregation of the sister chromosomes to prevent aneuploidy. Cancer cells often lack a functional G1 arrest and rely on G2 arrest for DNA damage responses. WEE1 kinase is an important regulator of the G2 checkpoint and is overexpressed in various cancer types. Inhibition of WEE1 is a promising strategy in cancer therapy in combination with DNA-damaging agents, especially when cancer cells harbor p53 mutations, as it causes mitotic catastrophy when DNA is not repaired during G2 arrest. Cancer cell response to WEE1 inhibition monotherapy has also been demonstrated in various types of cancer, including p53 wild-type cancers. We postulate that chromosomal instability can explain tumor response to WEE1 monotherapy. Therefore, chromosomal instability may need to be taken into account when determining the most effective strategy for the use of WEE1 inhibitors in cancer therapy.

Abstract Homologous recombination (HR) is a major mechanism of DNA repair, such that mutations in HR genes are associated with tumor susceptibility. We have used a recently adapted system for genome engineering to study HR mechanisms in mammalian cells. Clustered regularly-interspaced short palindromic repeats (CRISPR) are prokaryotic adaptive immune systems that specifically cleave nucleic acids of invading pathogens. The effector phase of CRISPR system in Staphylococcus pyogenes consists of two main components, the endonuclease Cas9 and RNA strands that dictate the recognition sequence cleaved by Cas9. The activity of Cas9 resides in 2 nucleolytic domains, HNH-like and RuvC1-like, such that mutating the active site of either of these domains turns Cas9 into a DNA nicking enzyme. Here we applied the commonly used DR-GFP HR reporter to investigate the repair of both DNA double-strand breaks (DSBs) and nicks induced by Cas9 in human cells. We show that a DSB, introduced by wild-type (wt) Cas9 and a guide RNA (gRNA) directed to the I-SceI site of the DR-GFP reporter, induces HR at levels similar to the I-SceI endonuclease, demonstrating the robustness of the system and confirming the potential of the CRISPR system as a genome engineering tool. Cas9 with mutations in either of the nucleolytic domains also stimulates HR, albeit at much lower levels, suggesting that a nick can also be repaired by HR. Stimulation of HR by DNA nicks is not reduced by PARP inhibition and has the same genetic requirements as DSB-induced HR. Given that SSBs are induced by endogenous DNA metabolic processes as well as by many anti-cancer treatments, our results may advance the understanding of mechanisms responsible for repair of these abundant DNA lesions. Citation Format: Lianne E.M. Vriend, Przemek M. Krawczyk, Maria Jasin. Induction of homologous recombination by DNA nicks. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C52.

Langerhans cells (LCs) lining the stratified epithelia and mucosal tissues are the first antigen presenting cells to encounter invading pathogens, such as viruses, bacteria and fungi. Fungal infections form a health threat especially in immuno-compromised individuals. LCs express C-type lectin Langerin that has specificity for mannose, fucose and GlcNAc structures. Little is known about the role of human Langerin in fungal infections. Our data show that Langerin interacts with both mannan and beta-glucan structures, common cell-wall carbohydrate structures of fungi. We have screened a large panel of fungi for recognition by human Langerin and, strikingly, we observed strong binding of Langerin to a variety of Candida and Saccharomyces species and Malassezia furfur, but very weak binding was observed to Cryptococcus gattii and Cryptococcus neoformans. Notably, Langerin is the primary fungal receptor on LCs, since the interaction of LCs with the different fungi was blocked by antibodies against Langerin. Langerin recognizes both mannose and beta-glucans present on fungal cell walls and our data demonstrate that Langerin is the major fungal pathogen receptor on human LCs that recognizes pathogenic and commensal fungi. Together these data may provide more insight in the role of LCs in fungal infections.