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Werner syndrome (WS), also known as adult progeria, is a rare autosomal recessive inherited progeroid syndrome characterised by multiple features consistent with accelerated ageing. This disease is associated with several rheumatic conditions such as early osteoarthritis and osteoporosis, sarcopenia, soft-tissue calcifications, gout, limb ulcers and scleroderma-like skin features. WS should be included in the differential diagnosis of systemic sclerosis. The diagnosis is clinical, and in 90% of cases, a genetic test reveals a pathogenic variant of the WRN gene.WRN encodes a member of the RecQ family of DNA helicases and has a role in DNA repair. 86 different pathological WRN mutations have been identified so far. Here we present a case report of a typical WS patient associated with a newly described genetic variant of the WRN gene.

In addition to the symptoms of aging, the main symptoms in Werner syndrome (WS), a hereditary premature aging disease, include calcification of subcutaneous tissue with solid pain and refractory skin ulcers. However, the mechanism of calcification in WS remains unclear. In this study, the histological analysis of the skin around the ulcer with calcification revealed an accumulation of calcium phosphate in the lymphatic vessels. Moreover, the morphological comparison with the lymphatic vessels in PAD patients with chronic skin ulcers demonstrated the ongoing lymphatic remodeling in WS patients because of the narrow luminal cross-sectional area (LA) of the lymphatic vessels but the increment of lymphatic microvessels density (MLVD). Additionally, fluorescence immunohistochemical analysis presented the cytoplasmic distribution and the accumulation of WRN proteins in endothelial cells on remodeling lymphatic vessels. In summary, these results point out a relationship between calcification in lymphatic vessels and the remodeling of lymphatic vessels and suggest the significance of the accumulation of WRN mutant proteins as an age-related change in WS patients. Thus, cytoplasmic accumulation of WRN protein can be an indicator of the decreasing drainage function of the lymphatic vessels and the increased risk of skin ulcers and calcification in the lymphatic vessels.

Brain tumors affect one-third of all children with cancer. Approximately 10% of children with cancer carry variants in cancer-predisposition genes. However, germline analyses in large cohorts of Asian children have not been reported. Thirty-eight Japanese patients with pediatric brain tumors were included in this study (19 boys, 19 girls). DNA was extracted from the patients’ peripheral blood, and cancer-associated genes were analyzed using targeted resequencing. Rare variants with allele frequencies CHEK2 and FANCI; 2 frameshift deletions in SMARCB1 and PTCH1; and 3 missense variants of TSC1, WRN, and MLH1. The median age at diagnosis was 9.1 years, and three of the 7 patients had a family history of cancer. One patient diagnosed with basal cell nevus syndrome, also called Gorlin syndrome, developed a second neoplasm, and another with an SMARCB1 variant and an atypical teratoid/rhabdoid tumor developed a thyroid adenomatous nodule. This is the first cancer-related germline analysis with detailed clinical information reported in Japanese children with brain tumors. The prevalence was almost equivalent to that in white children.

One of the goals of precision medicine is to uncover selective vulnerabilities in various cancers. A notable success has been the development of PARP inhibitors for the treatment of breast and ovarian cancers with mutations in BRCA genes. Only two years ago, it was discovered that cancers with microsatellite instability (MSI) were selectively dependent on the RecQ DNA helicase WRN. Subsequently, the molecular mechanism underlying WRN dependency in MSI cancers was uncovered. Here, we review how these developments have led to a promising new drug target in MSI cancers.

Synthetic lethality is a powerful approach for targeting oncogenic drivers in cancer. Recent studies revealed that cancer cells with microsatellite instability (MSI) require Werner (WRN) helicase for survival; however, the underlying mechanism remains unclear. In this study, we found that WRN depletion strongly induced p53 and its downstream apoptotic target PUMA in MSI colorectal cancer (CRC) cells. p53 or PUMA deletion abolished apoptosis induced by WRN depletion in MSI CRC cells. Importantly, correction of MSI abrogated the activation of p53/PUMA and cell killing, while induction of MSI led to sensitivity in isogenic CRC cells. Rare p53-mutant MSI CRC cells are resistant to WRN depletion due to lack of PUMA induction, which could be restored by wildtype (WT) p53 knock in or reconstitution. WRN depletion or treatment with the RecQ helicase inhibitor ML216 suppressed in vitro and in vivo growth of MSI CRCs in a p53/PUMA-dependent manner. ML216 treatment was efficacious in MSI CRC patient-derived xenografts. Interestingly, p53 gene remains WT in the majority of MSI CRCs. These results indicate a critical role of p53/PUMA-mediated apoptosis in the vulnerability of MSI CRCs to WRN loss, and support WRN as a promising therapeutic target in p53 -WT MSI CRCs.

Exonucleases are often found associated with polymerase or helicase domains in the same enzyme or can function as autonomous entities to maintain genome stability. Here, we uncovered Chaetomium thermophilum RecQ family proteins that also have exonuclease activity in addition to their main helicase function. The novel exonuclease activity is separate from the helical core domain and coexists with the latter two enzymatic activities on the same polypeptide. The CtRecQ

Werner Syndrome (WS) is an autosomal recessive disorder characterized by premature aging due to mutations of the WRN gene. A classical sign in WS patients is short stature, but the underlying mechanisms are not well understood. Here we report that WRN is indispensable for chondrogenesis, which is the engine driving the elongation of bones and determines height. Zebrafish lacking wrn exhibit impairment of bone growth and have shorter body stature. We pinpoint the function of WRN to its helicase domain. We identify short-stature homeobox (SHOX) as a crucial and direct target of WRN and find that the WRN helicase core regulates the transcriptional expression of SHOX via unwinding G-quadruplexes. Consistent with this, shox−/− zebrafish exhibit impaired bone growth, while genetic overexpression of SHOX or shox expression rescues the bone developmental deficiency induced in WRN/wrn-null mutants both in vitro and in vivo. Collectively, we have identified a previously unknown function of WRN in regulating bone development and growth through the transcriptional regulation of SHOX via the WRN helicase domain, thus illuminating a possible approach for new therapeutic strategies.

Helicases, DNA translocases, nucleases and DNA-binding proteins play integral roles in protecting replication forks in human cells. Perturbations to replication fork dynamics can be caused by genetic loss of key factor(s) or exposure to replication stress inducing agents that perturb the nucleotide pool, stabilize unusual DNA secondary structures, or inhibit protein function (typically catalytic activity performed by a DNA polymerase, nuclease or helicase). DNA fiber analysis is a highly resourceful and facile experimental approach to study the molecular dynamics of replication forks in living cells. In this chapter, we provide a detailed list of reagents, equipment and experimental strategies to perform DNA fiber experiments. We have utilized these approaches to characterize the role of the Werner syndrome helicase (WRN) to protect replication forks in cells that are deficient in the tumor suppressor and genome stability factor BRCA2.

Targeted therapies, chemotherapy, and immunotherapy are used to treat patients with mismatch repair–deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer. The clinical effectiveness of targeted therapy and chemotherapy is limited by resistance and drug toxicities, and about half of patients receiving immunotherapy have disease that is refractory to immune checkpoint inhibitors. Loss of Werner syndrome ATP-dependent helicase (WRN) is a synthetic lethality in dMMR/MSI-H cells. To inform the development of WRN as a therapeutic target, we performed WRN knockout or knockdown in 60 heterogeneous dMMR colorectal cancer preclinical models, demonstrating that WRN dependency is an almost universal feature and a robust marker for patient selection. Furthermore, models of resistance to clinically relevant targeted therapy, chemotherapy, and immunotherapy retain WRN dependency. These data show the potential of therapeutically targeting WRN in patients with dMMR/MSI-H colorectal cancer and support WRN as a therapeutic option for patients with dMMR/MSI-H cancers refractory to current treatment strategies. Significance: We found that a large, diverse set of dMMR/MSI-H colorectal cancer preclinical models, including models of treatment-refractory disease, are WRN-dependent. Our results support WRN as a promising synthetic-lethal target in dMMR/MSI-H colorectal cancer tumors as a monotherapy or in combination with targeted agents, chemotherapy, or immunotherapy. This article is highlighted in the In This Issue feature, p. 1861

Werner syndrome (WRN) is a rare progressive genetic disorder, caused by functional defects in WRN protein and RecQ4L DNA helicase. Acceleration of the aging process is initiated at puberty and the expected life span is approximately the late 50 s. However, a Wrn-deficient mouse model does not show premature aging phenotypes or a short life span, implying that aging processes differ greatly between humans and mice. Gene expression analysis of WRN cells reveals very similar results to gene expression analysis of Hutchinson Gilford progeria syndrome (HGPS) cells, suggesting that these human progeroid syndromes share a common pathological mechanism. Here we show that WRN cells also express progerin, an abnormal variant of the lamin A protein. In addition, we reveal that duplicated sequences of human WRN (hWRN) from exon 9 to exon 10, which differ from the sequence of mouse WRN (mWRN), are a natural inhibitor of progerin. Overexpression of hWRN reduced progerin expression and aging features in HGPS cells. Furthermore, the elimination of progerin by siRNA or a progerin-inhibitor (SLC-D011 also called progerinin) can ameliorate senescence phenotypes in WRN fibroblasts and cardiomyocytes, derived from WRN-iPSCs. These results suggest that progerin, which easily accumulates under WRN-deficient conditions, can lead to premature aging in WRN and that this effect can be prevented by SLC-D011.

Werner syndrome is a premature ageing disorder caused by biallelic variants in the WRN gene. WRN encodes a dual DNA helicase/exonuclease enzyme. Molecular diagnosis is commonly only made at a late disease stage in the third or fourth decade, when cardinal features have become apparent. We describe a 28 year-old woman who presented with early onset diabetes associated with partial lipodystrophy, severe dyslipidaemia and rapidly progressive liver fibrosis related to non-alcoholic steatohepatitis in the absence of progeroid features. Werner syndrome was diagnosed by trio exome analysis, which revealed compound heterozygous WRN mutations: the known variant c.1290_1293del (p.Asn430Lysfs*7) and the novel intronic splice site variant c.2732+5G>A. cDNA analysis demonstrated this to lead to in-frame skipping of exon 22, predicted to delete most of the zinc binding region of the helicase domain. We suggest that including the WRN gene in genetic analysis of early onset diabetes, lipodystrophy or dyslipidaemia would allow for the opportunity to diagnose some cases of Werner syndrome long before clinical criteria are met, thereby allowing early implementation of important primary prevention interventions.

Background Werner syndrome (WS) is an autosomal recessive premature ageing disease that causes accelerated ageing-like symptoms after puberty. Previous studies conducted in the late 2000s reported that malignant neoplasms and atherosclerotic diseases were the two leading causes of death, with life expectancies in the mid-50 s. However, the recent lifespan and cause of death in patients with WS remain unclear. Objective To clarify the latest lifespan and causes of death in patients with WS. Method We conducted a questionnaire-based survey in 2020 among the primary doctors of WS patients who were identified in previous nationwide surveys in Japan and clarified the following: the age of WS patients (age of death, if the patient had already died), sex, and cause of death. Patients who died in 2010 or earlier were excluded from the analysis. Results A total of 123 living patients were identified at the time of the survey in 2020. Fourteen WS patients died between 2011 and 2020, with a mean age of 59.0 ± 8.9 years (mean ± SD). The most common cause of death was non-epithelial tumours, accounting for eight deaths, while no patient died of atherosclerotic diseases. Conclusions Compared to previous studies, this study suggests that the lifespan of patients with WS has been extended. Although there were no deaths due to atherosclerotic diseases, non-epithelial tumours were still the leading cause of death. Further development of screening and treatment methods for these tumours is required.

Hereditary breast and ovarian cancers are frequently attributed to germline mutations in the tumor suppressor genes BRCA1 and BRCA2. BRCA1/2 act to repair double-strand breaks (DSBs) and suppress the demise of unstable replication forks. Our work elucidated a dynamic interplay between BRCA2 and the WRN DNA helicase/exonuclease defective in the premature aging disorder Werner syndrome. WRN and BRCA2 participate in complementary pathways to stabilize replication forks in cancer cells, allowing them to proliferate. Whether the functional overlap of WRN and BRCA2 is relevant to replication at gaps between newly synthesized DNA fragments, protection of telomeres, and/or metabolism of secondary DNA structures remain to be determined. Advances in understanding the mechanisms elicited during replication stress have prompted the community to reconsider avenues for cancer therapy. Insights from studies of PARP or topoisomerase inhibitors provide working models for the investigation of WRN's mechanism of action. We discuss these topics, focusing on the implications of the WRN-BRCA2 genetic interaction under conditions of replication stress.

Mutation in Werner (WRN) RECQL helicase is associated with premature aging syndrome (Werner syndrome, WS) and predisposition to multiple cancers. In patients with solid cancers, deficiency of the WRN RECQL helicase is paradoxically associated with enhanced overall survival in response to treatment with TOP1 inhibitors, which stabilize pathological TOP1-DNA-covalent-complexes (TOP1cc) on the genome. However, the underlying mechanism of WRN in development of chemoresistance to TOP1 inhibitors is not yet explored. Our whole-genome transcriptomic analysis for ~25,000 genes showed robust activation of NF-κB-dependent prosurvival genes in response to TOP1cc. CRISPR-Cas9 knockout, shRNA silencing, and under-expression of WRN confer high-sensitivity of multiple cancers to TOP1 inhibitor. We demonstrated that WRN orchestrates TOP1cc repair through proteasome-dependent and proteasome-independent process, unleashing robust ssDNA generation. This in turn ensues signal transduction for CHK1 mediated NF-κB-activation through IκBα-degradation and nuclear localization of p65 protein. Intriguingly, our site-directed mutagenesis and rescue experiments revealed that neither RECQL-helicase nor DNA-exonuclease enzyme activity of WRN (WRN

For the purpose of examining the characteristics of dyslipidemia and fatty liver in patients with Werner syndrome in Japan in recent years, we searched all case reports of Japanese Werner syndrome reported on Medical Online and PubMed since 1996, and collected and examined the data and clinical features described in these reports. In addition, as there are few descriptions of treatment methods in these reports from Medical Online and PubMed, we analyzed 12 cases for which detailed data on treatment methods are available at Chiba University. Geriatr Gerontol Int 2021; 21: 133-138.

Background Apoptosis and oxidative stress are the main etiology of age related cataract (ARC). This article aims to investigate the role of WRN in lens epithelial cells (LECs). Methods We estimated the methylation level of WRN in anterior lens capsule tissues of ARC patients. SRA01/04 (LECs) cells were treated with H2O2 or combined with 5-aza-2-deoxycytidine (5-Aza-CdR) or chloroquine. CCK8 and flow cytometry were performed to explore proliferation and apoptosis. The content of ROS was detected by fluorescent probe DCFH-DA. The gene and protein expression was assessed by quantitative real-time PCR or western blot. Results WRN was down-regulated and the methylation level of WRN was increased in the anterior lens capsule tissues. WRN overexpression and 5-Aza-CdR enhanced proliferation and repressed apoptosis and oxidative stress of SRA01/04 cells. 5-Aza-CdR enhanced WRN expression. WRN knockdown inhibited proliferation and promoted apoptosis and oxidative stress of SRA01/04 cells, which was rescued by 5-Aza-CdR. WRN overexpression and 5-Aza-CdR repressed ATM/p53 signaling pathway. Furthermore, chloroquine inhibited proliferation and promoted apoptosis and oxidative stress of SRA01/04 cells by activating ATM/p53 signaling pathway. The influence conferred by chloroquine was abolished by WRN overexpression. Conclusion Our study reveals that DNA methylation mediated WRN inhibits apoptosis and oxidative stress of human LECs through ATM/p53 signaling pathway.

Maintenance of genome stability is crucial for cell survival and relies on accurate DNA replication. However, replication fork progression is under constant attack from different exogenous and endogenous factors that can give rise to replication stress, a source of genomic instability and a notable hallmark of pre-cancerous and cancerous cells. Notably, one of the major natural threats for DNA replication is transcription. Encounters or conflicts between replication and transcription are unavoidable, as they compete for the same DNA template, so that collisions occur quite frequently. The main harmful transcription-associated structures are R-loops. These are DNA structures consisting of a DNA–RNA hybrid and a displaced single-stranded DNA, which play important physiological roles. However, if their homeostasis is altered, they become a potent source of replication stress and genome instability giving rise to several human diseases, including cancer. To combat the deleterious consequences of pathological R-loop persistence, cells have evolved multiple mechanisms, and an ever growing number of replication fork protection factors have been implicated in preventing/removing these harmful structures; however, many others are perhaps still unknown. In this review, we report the current knowledge on how aberrant R-loops affect genome integrity and how they are handled, and we discuss our recent findings on the role played by two fork protection factors, the Werner syndrome protein (WRN) and the Werner helicase-interacting protein 1 (WRNIP1) in response to R-loop-induced genome instability.

The tumor suppressor BRCA2 protects stalled forks from degradation to maintain genome stability. However, the molecular mechanism(s) whereby unprotected forks are stabilized remains to be fully characterized. Here, we demonstrate that WRN helicase ensures efficient restart and limits excessive degradation of stalled forks in BRCA2-deficient cancer cells. In vitro, WRN ATPase/helicase catalyzes fork restoration and curtails MRE11 nuclease activity on regressed forks. We show that WRN helicase inhibitor traps WRN on chromatin leading to rapid fork stalling and nucleolytic degradation of unprotected forks by MRE11, resulting in MUS81-dependent double-strand breaks, elevated non-homologous end-joining and chromosomal instability. WRN helicase inhibition reduces viability of BRCA2-deficient cells and potentiates cytotoxicity of a poly (ADP)ribose polymerase (PARP) inhibitor. Furthermore, BRCA2-deficient xenograft tumors in mice exhibited increased DNA damage and growth inhibition when treated with WRN helicase inhibitor. This work provides mechanistic insight into stalled fork stabilization by WRN helicase when BRCA2 is deficient., The tumor suppressor BRCA2 protects stalled DNA replication forks from unrestrained degradation; however the mechanism whereby unprotected stalled forks are preserved and restarted has remained elusive. Here the authors show that the WRN helicase promotes stalled fork recovery and limits fork hyper-degradation in the absence of BRCA2 protection.

As organisms age, their resistance to stress decreases while their risk of disease increases. This can be shown in patients with Werner syndrome (WS), which is a genetic disease characterized by accelerated aging along with increased risk of cancer and metabolic disease. WS is caused by mutations in WRN, a gene involved in DNA replication and repair. Recent research has shown that WRN mutations contribute to multiple hallmarks of aging including genomic instability, telomere attrition, and mitochondrial dysfunction. However, questions remain regarding the onset and effect of stress on early aging. We used a fly model of WS (WRNexoΔ) to investigate stress response during different life stages and found that stress sensitivity varies according to age and stressor. While larvae and young WRNexoΔ adults are not sensitive to exogenous oxidative stress, high antioxidant activity suggests high levels of endogenous oxidative stress. WRNexoΔ adults are sensitive to stress caused by elevated temperature and starvation suggesting abnormalities in energy storage and a possible link to metabolic dysfunction in WS patients. We also observed higher levels of sleep in aged WRNexoΔ adults suggesting an additional adaptive mechanism to protect against age-related stress. We suggest that stress response in WRNexoΔ is multifaceted and evokes a systemic physiological response to protect against cellular damage. These data further validate WRNexoΔ flies as a WS model with which to study mechanisms of early aging and provide a foundation for development of treatments for WS and similar diseases.

A rare and autosomal recessive premature aging disorder, Werner syndrome (WS) is characterized by the early onset of aging-associated diseases, including shortening stature, alopecia, bilateral cataracts, skin ulcers, diabetes, osteoporosis, arteriosclerosis, and chromosomal instability, as well as cancer predisposition. WRN, the gene responsible for WS, encodes DNA helicase with a 3′ to 5′ exonuclease activity, and numerous studies have revealed that WRN helicase is involved in the maintenance of chromosome stability through actions in DNA, e.g., DNA replication, repair, recombination, and epigenetic regulation via interaction with DNA repair factors, telomere-binding proteins, histone modification enzymes, and other DNA metabolic factors. However, although these efforts have elucidated the cellular functions of the helicase in cell lines, they have not been linked to the treatment of the disease. Life expectancy has improved for WS patients over the past three decades, and it is hoped that a fundamental treatment for the disease will be developed. Disease-specific induced pluripotent stem (iPS) cells have been established, and these are expected to be used in drug discovery and regenerative medicine for WS patients. In this article, we review trends in research to date and present some perspectives on WS research with regard to the application of pluripotent stem cells. Furthermore, the elucidation of disease mechanisms and drug discovery utilizing the vast amount of scientific data accumulated to date will be discussed.

Werner syndrome (WS) is an accelerated aging disorder characterized by genomic instability, which is caused by WRN protein deficiency. WRN participates in DNA metabolism including DNA repair. In a previous report, we showed that WRN protein is recruited to laser‐induced DNA double‐strand break (DSB) sites during various stages of the cell cycle with similar intensities, supporting that WRN participates in both non‐homologous end joining (NHEJ) and homologous recombination (HR). Here, we demonstrate that the phosphorylation of WRN by CDK2 on serine residue 426 is critical for WRN to make its DSB repair pathway choice between NHEJ and HR. Cells expressing WRN engineered to mimic the unphosphorylated or phosphorylation state at serine 426 showed abnormal DSB recruitment, altered RPA interaction, strand annealing, and DSB repair activities. The CDK2 phosphorylation on serine 426 stabilizes WRN’s affinity for RPA, likely increasing its long‐range resection at the end of DNA strands, which is a crucial step for HR. Collectively, the data shown here demonstrate that a CDK2‐dependent phosphorylation of WRN regulates DSB repair pathway choice and cell cycle participation., When DSBs occurs in the G1 phase of the cell cycle, the KU70/80 complex interacts directly with WRN and stimulates WRN's exonuclease activity. Stimulated by KU70/80, DNA‐PKcs also regulates WRN's enzymatic activities. Finally, fully activated WRN utilizes its nuclease activity to create DNA ends suitable for XRCC4‐DNA ligase IV complex mediated ligation. When cells progress into S phase of the cell cycle, CDK2 becomes active via CycE, and phosphorylates WRN on S426. S426 phosphorylated WRN has increased affinity for RPA, thereby promoting end resection and stabilization of the resected single‐stranded DNA. Also, S426 phosphorylation enhances WRN's strand annealing activity to promote strand annealing activity to form D‐loop formation for efficient HR.

Generalized lipodystrophy (GL) syndromes are a group of rare heterogenous disorders, characterized by total subcutaneous fat loss. The frequency of GL is currently assessed as approximately 0,23 cases per million of the population, in Europe - as 0,96 cases per million of the population. They can be congenital (CGL) or acquired (AGL) depending on the etiology and the time of the onset of fat loss. Both CGL and AGL are often associated with different metabolic complications, such as hypertriglyceridemia, insulin resistance and lipoatrophic diabetes mellitus, metabolically associated FLD, arterial hypertension, proteinuria, reproductive system disorders. In this review we aimed to summarize the information on all forms of generalized lipodystrophy, especially the ones of genetic etiology, their clinical manifestations and complications, the perspectives for diagnostics, treatment and further research.

G:C sites distant from 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) are frequently mutated when the lesion-bearing plasmid DNA is replicated in human cells with reduced Werner syndrome (WRN) protein. To detect the untargeted mutations preferentially, the oxidised guanine base was placed downstream of the reporter supF gene and the plasmid DNA was introduced into WRN-knockdown cells. The total mutant frequency seemed higher in the WRN-knockdown cells as compared to the control cells. Mutation analyses revealed that substitution mutations occurred at the G:C pairs of 5′-GpA-3′/5′-TpC-3′ sites, the preferred sequence for the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3)-family cytosine deaminases, in the supF gene in both control and knockdown cells. These mutations were observed more frequently at G sites than C sites on the DNA strand where the GO base was originally located. This tendency was promoted by the knockdown of the WRN protein. The present results imply the possible involvement of APOBEC3-family cytosine deaminases in the action-at-a-distance (untargeted) mutations at G:C (or G) sites induced by GO and in cancer initiation by oxidative stress.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

Werner syndrome (WS) is a cancer-prone disease caused by deficiency of Werner protein (WRN). WRN maintains genome integrity by promoting replication-fork stability after various forms of replication stress. Under mild replication stress, WS cells show impaired ATR-mediated CHK1 activation. However, it remains unclear if WS cells elicit other repair pathway. We demonstrate that loss of WRN leads to enhanced ATM phosphorylation upon prolonged exposure to aphidicolin, a specific inhibitor of DNA polymerases, resulting in CHK1 activation. Moreover, we find that loss of WRN sensitises cells to replication-transcription collisions and promotes accumulation of R-loops, which undergo XPG-dependent cleavage responsible for ATM signalling activation. Importantly, we observe that ATM pathway limits chromosomal instability in WS cells. Finally, we prove that, in WS cells, genomic instability enhanced upon chemical inhibition of ATM kinase activity is counteracted by direct or indirect suppression of R-loop formation or by XPG abrogation. Together, these findings suggest a potential role of WRN as regulator of R-loop-associated genomic instability, strengthening the notion that conflicts between replication and transcription can affect DNA replication, leading to human disease and cancer.

MDM2 (Murine double minute 2) acts as a key repressor for p53-mediated tumor-suppressor functions, which includes cellular senescence. We found that MDM2 can promote cellular senescence by modulating WRN stability. Werner syndrome (WS), caused by mutations of the WRN gene, is an autosomal recessive disease, which is characterized by premature aging. Loss of WRN function induces cellular senescence in human cancer cells. Here, we found that MDM2 acts as an E3 ligase for WRN protein. MDM2 interacts with WRN both in vivo and in vitro. MDM2 induces ubiquitination of WRN and dramatically downregulates the levels of WRN protein in human cells. During DNA damage response, WRN is translocated to the nucleoplasm to facilitate its DNA repair functions; however, it is degraded by the MDM2-mediated ubiquitination pathway. Moreover, the senescent phenotype induced by DNA damage reagents, such as Etoposide, is at least in part mediated by MDM2-dependent WRN degradation as it can be significantly attenuated by ectopic expression of WRN. These results show that MDM2 is critically involved in regulating WRN function via ubiquitin-dependent degradation and reveal an unexpected role of MDM2 in promoting cellular senescence through a p53-independent manner.

Adult progeria Werner syndrome (WS), a rare autosomal recessive disorder, is characterized by accelerated aging symptoms after puberty. The causative gene, WRN, is a member of the RecQ DNA helicase family and is predominantly involved in DNA replication, repair, and telomere maintenance. Here, we report the generation of iPS cells from a patient with WS and correction of the WRN gene by the CRISPR/Cas9-mediated method. These iPSC lines would be a valuable resource for deciphering the pathogenesis of WS.

The nucleolus is a nuclear structure composed of ribosomal DNA (rDNA), and functions as a site for rRNA synthesis and processing. The rDNA is guanine-rich and prone to form G-quadruplex (G4), a secondary structure of DNA. We have recently found that HERC2, an HECT ubiquitin ligase, promotes BLM and WRN RecQ DNA helicases to resolve the G4 structure. Here, we report the role of HERC2 in the regulation of nucleolar localization of the helicases. Furthermore, HERC2 inactivation enhances the effects of CX-5461, an inhibitor of RNA polymerase I (Pol I)-mediated transcription of rRNA with an intrinsic G4-stabilizing activity. HERC2 depletion or homozygous deletion of the C-terminal HECT domain of HERC2 prevented the nucleolar localization of BLM and WRN, and inhibited relocalization of BLM to replication stress-induced nuclear RPA foci. HERC2 colocalized with fibrillarin and Pol I subunit RPA194, both of which are required for rRNA transcription. The HERC2 dysfunction enhanced the suppression of pre-rRNA transcription by CX-5461. These results suggest the effect of HERC2 status on the functions of BLM and WRN on rRNA transcription in the nucleolus. Since HERC2 is downregulated in numerous cancers, this effect may be clinically relevant considering the beneficial effects of CX-5461 in cancer treatments.

RecQ DNA helicases are a conserved protein family found in bacteria, fungus, plants, and animals. These helicases play important roles in multiple cellular functions, including DNA replication, transcription, DNA repair, and telomere maintenance. Humans have five RecQ helicases: RECQL1, Bloom syndrome protein (BLM), Werner syndrome helicase (WRN), RECQL4, and RECQL5. Defects in BLM and WRN cause autosomal disorders: Bloom syndrome (BS) and Werner syndrome (WS), respectively. Mutations in RECQL4 are associated with three genetic disorders, Rothmund–Thomson syndrome (RTS), Baller–Gerold syndrome (BGS), and RAPADILINO syndrome. Although no genetic disorders have been reported due to loss of RECQL1 or RECQL5, dysfunction of either gene is associated with tumorigenesis. Multiple genetically independent pathways have evolved that mediate the repair of DNA double-strand break (DSB), and RecQ helicases play pivotal roles in each of them. The importance of DSB repair is supported by the observations that defective DSB repair can cause chromosomal aberrations, genomic instability, senescence, or cell death, which ultimately can lead to premature aging, neurodegeneration, or tumorigenesis. In this review, we will introduce the human RecQ helicase family, describe in detail their roles in DSB repair, and provide relevance between the dysfunction of RecQ helicases and human diseases.

Significance: Werner syndrome (WS) is a rare autosomal recessive malady typified by a pro-oxidant/proinflammatory status, genetic instability, and by the early onset of numerous age-associated illn...

International audience; Scope Cholesterol bioavailability displays a high interindividual variability, partly due to genetic factors. Existing studies have focused on single nucleotide polymorphisms (SNPs) analyzed individually, which only explained a minor fraction of the variability of this complex phenotype. The aim is to identify a combination of SNPs associated with a significant part of the variability in cholesterol bioavailability. Methods and results Thirty-nine healthy adult males are given a standard test snack containing 80 mg heptadeuterated (D7) cholesterol. The plasma D7-cholesterol concentration is measured at equilibrium 40 h after test snack intake. The D7-cholesterol response (D7-cholesterol/total cholesterol concentration) exhibits a relatively high interindividual variability (CV = 32%). The association of exonic SNPs in candidate genes (188 genes involved in or related to cholesterol metabolism) with the plasma D7-cholesterol response is assessed by univariate statistics followed by partial least squares regression. A significant model (p-value after cross-validation ANOVA = 1.64 x 10(-7)) that includes 8 SNPs (SOAT2-rs9658625,DNAH11-rs11768670,LIPC-rs690,MVK-rs2287218,GPAM-rs10787428,APOE-rs7412,CBS-rs234706, andWRN-rs1801196) explains 59.7% of the variance in cholesterol bioavailability (adjustedR(2)). Conclusion Here a combination of SNPs is significantly associated with the variability in dietary cholesterol bioavailability in healthy adult males.

Werner syndrome, also called adult progeria, is a heritable autosomal recessive human disorder characterized by the premature onset of numerous age-related diseases including juvenile cataracts, dyslipidemia, diabetes mellitus (DM), osteoporosis, atherosclerosis, and cancer. Werner syndrome is a segmental progeroid syndrome whose presentation resembles accelerated aging. The most common causes of death for WS patients are atherosclerosis and cancer. A 40-year-old female presented with short stature, bird-like facies, canities with alopecia, scleroderma-like skin changes, and non-healing foot ulcers. The patient reported a history of delayed puberty, abortion, hypertriglyceridemia, and juvenile cataracts. A clinical diagnosis of WS was made and subsequently confirmed. We discovered two WRN gene mutations in the patient, Variant 1 was the most common WRN mutation, nonsense mutation (c.1105C>T:p.R369Ter) in exon 9, which caused a premature termination codon (PTC) at position 369. Variant 2 was a frameshift mutation (c.1134delA:p.E379KfsTer5) in exon 9, which caused a PTC at position 383 and has no published reports describing. Patients with WS can show a wide variety of clinical and biological manifestations in endocrine-metabolic systems (DM, thyroid dysfunction, and hyperlipidemia). Doctors must be cognizant of early manifestations of WS and treatment options.

Werner (WRN) expression is epigenetically downregulated in various tumors. It is imperative to understand differential repair process in WRN-proficient and WRN-deficient cancers to find pharmacological targets for radio-sensitization of WRN-deficient cancer. In the current investigation, we showed that pharmacological inhibition of CHK1 mediated homologous recombination repair (HRR), but not non-homologous end joining (NHEJ) repair, can causes hyper-radiosensitization of WRN-deficient cancers. This was confirmed in cancer cell lines of different tissue origin (osteosarcoma, colon adenocarcinoma and melanoma) with WRN silencing and overexpression. We established that WRN-depleted cells are dependent on a critical but compromised CHK1-mediated HRR-pathway for repairing ionizing radiation (IR) induced DSBs for their survival. Mechanistically, we unraveled a new finding that the MRE11, CTIP and WRN proteins are largely responsible for resections of late and persistent DSBs. In response to IR-treatment, MRE11 and CTIP-positively and WRN-negatively regulate p38-MAPK reactivation in a CHK1-dependent manner. A degradation resistant WRN protein, mutated at serine 1141, abrogates p38-MAPK activation. We also showed that CHK1-p38-MAPK axis plays important role in RAD51 mediated HRR in WRN-silenced cells. Like CHK1 inhibition, pharmacological-inhibition of p38-MAPK also hyper-radiosensitizes WRN-depleted cells by targeting HR-pathway. Combination treatment of CHK1-inhibitor (currently under various clinical trials) and IR exhibited a strong synergy against WRN-deficient melanoma tumor in vivo. Taken together, our findings suggest that pharmacological targeting of CHK1-p38-MAPK mediated HRR is an attractive strategy for enhancing therapeutic response of radiation treatment of cancer.