Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model.

The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.

WRN rescues replication forks compromised by a BRCA2 deficiency: Predictions for how inhibition of a helicase that suppresses premature aging tilts the balance to fork demise and chromosomal instability in cancer.

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.

Paradoxical role of the major DNA repair protein, OGG1, in action-at-a-distance mutation induction by 8-oxo-7,8-dihydroguanine.

Oxidatively damaged bases induce mutations and are involved in cancer initiation. 8-Oxo-7,8-dihydroguanine (G°, 8-hydroxyguanine) is an abundant oxidized base that induces targeted G:C→T:A transversions in human cells, as well as untargeted base substitution (action-at-a-distance) mutations of the G bases of 5'-GpA-3' dinucleotides. The action-at-a-distance mutations become more frequent than the targeted transversions when the amount of Werner syndrome (WRN) protein is decreased. In this study, OGG1, the major DNA glycosylase for the damaged base, and WRN were knocked down in isolation and in combination in human U2OS cells, and a shuttle plasmid carrying G° was introduced into the knockdown cells. Interestingly, fewer action-at-a-distance mutations were observed in the WRN plus OGG1 double knockdown cells, as compared to the WRN single knockdown cells. These results indicated the paradoxical role of OGG1, as an accelerator of the action-at-a-distance mutations by the oxidized guanine base.

CDK2 phosphorylation of Werner protein (WRN) contributes to WRN's DNA double-strand break repair pathway choice.

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.

Action-at-a-distance mutations at 5'-GpA-3' sites induced by oxidised guanine in WRN-knockdown cells.

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.

Severe metabolic disorders coexisting with Werner syndrome: a case report.

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 Syndrome Protein Expression in Breast Cancer.

INTRODUCTION: Werner protein (WRN) plays an important role in DNA repair, replication, transcription, and consequently genomic stability via its DNA-helicase and exonuclease activity. Loss of function of WRN is associated with Werner syndrome (WS), which is characterized by premature aging and cancer predisposition. Malignancies that are commonly linked to WS are thyroid carcinoma, melanoma, breast cancer, meningioma, and soft tissue and bone sarcomas. Currently, the clinicopathologic significance of WRN in breast cancer is largely unknown. PATIENTS AND METHODS: We investigated the clinicopathologic and prognostic significance of WRN protein expression in a cohort of clinically annotated series of sporadic (n = 1650) and BRCA-mutated (n = 75) invasive breast cancers. We correlated WRN protein expression to clinicopathologic characteristics, DNA repair protein expression, and survival outcomes. RESULTS: There is strong evidence of association between low nuclear and cytoplasmic WRN co-expression and low levels of KU70/KU80, DNA-PK, DNA Pol-B, CKD18, cytoplasmic RECQL4, and nuclear BLM protein expression (adjusted P-values < .05). Tumors with low nuclear or cytoplasmic WRN expression have worse overall breast cancer-specific survival (BCSS) (adjusted P-values < .05). In topoisomerase I overexpressed tumors, low WRN nuclear expression was associated with poor BCSS (P-value < .05). In BRCA-mutated tumors, low WRN cytoplasmic expression conferred shortest BCSS (P < .05). CONCLUSIONS: Low WRN protein expression is associated with poor BCSS in patients with breast cancer. This can be used to optimize the risk stratification for personalized treatment.

WRN modulates translation by influencing nuclear mRNA export in HeLa cancer cells.

BACKGROUND: The Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation. RESULTS: To identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN interacts with mRNA and the Nuclear RNA Export Factor 1 (NXF1). CONCLUSIONS: Our findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.

Genetic correction of Werner syndrome gene reveals impaired pro-angiogenic function and HGF insufficiency in mesenchymal stem cells.

WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN+/+ mesenchymal stem cells (MSCs) exhibited improved pro-angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN-/- MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN-/- MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro-angiogenic function of WS-MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.

RAD51 and mitotic function of mus81 are essential for recovery from low-dose of camptothecin in the absence of the WRN exonuclease.

Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.

MDM2-mediated degradation of WRN promotes cellular senescence in a p53-independent manner.

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.

Mutations induced by 8-oxo-7,8-dihydroguanine in WRN- and DNA polymerase λ-double knockdown cells.

Reactive oxygen species generate 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine), which induces G:C→T:A transversion mutations. The knockdowns of the protein responsible for Werner syndrome (WRN), a cancer-associated DNA helicase, and DNA polymerase (pol) λ, a WRN-interacting DNA pol, cause untargeted base-substitution mutations (action-at-a-distance mutations). To examine the consequences of the dual reductions of WRN and pol λ for the mutations caused by GO, siRNAs against both proteins were introduced into human U2OS cells. A replicable plasmid DNA with the oxidised nucleobase in a unique position of the supF gene was then introduced into the double knockdown cells. The amplified DNA recovered from the cells was used to transform a bacterial indicator strain. The mutant frequency and the subsequent sequence analysis revealed that the double knockdown additively promoted the G:C→T:A substitution at the GO position and increased the action-at-a-distance mutations to a level similar to that of the single WRN knockdown. Thus, WRN and DNA pol λ seem to suppress the targeted G:C→T:A mutation at least in part independently and reduce the untargeted mutations via an identical pathway.

Werner syndrome (WRN) gene variants and their association with altered function and age-associated diseases.

Werner syndrome (WS) is a heritable autosomal recessive human disorder characterized by the premature onset of several age-associated pathologies including cancer. The protein defective in WS patients, WRN, is encoded by a member of the human RECQ gene family that contains both a DNA exonuclease and a helicase domain. WRN has been shown to participate in several DNA metabolic pathways including DNA replication, recombination and repair, as well as telomere maintenance and transcription modulation. Here we review base pair-level genetic variation that has been documented in WRN, with an emphasis on non-synonymous coding single nucleotide polymorphisms (SNPs) and their associations with anthropomorphic features, longevity and disease risk. These associations have been challenging to identify, as many reported WRN SNP associations appear to be further conditioned upon ethnic, age, gender or other environmental co-variables. The WRN variant phenotypic associations identified to date are intriguing, and several are of clear clinical import. Consequently, it will be important to extend these initial associations and to identify the mechanisms and conditions under which specific WRN variants may compromise WRN function to drive cellular and organismal phenotypes as well as disease risk.

WRN regulates pathway choice between classical and alternative non-homologous end joining.

Werner syndrome (WS) is an accelerated ageing disorder with genomic instability caused by WRN protein deficiency. Many features seen in WS can be explained by the diverse functions of WRN in DNA metabolism. However, the origin of the large genomic deletions and telomere fusions are not yet understood. Here, we report that WRN regulates the pathway choice between classical (c)- and alternative (alt)-nonhomologous end joining (NHEJ) during DNA double-strand break (DSB) repair. It promotes c-NHEJ via helicase and exonuclease activities and inhibits alt-NHEJ using non-enzymatic functions. When WRN is recruited to the DSBs it suppresses the recruitment of MRE11 and CtIP, and protects the DSBs from 5' end resection. Moreover, knockdown of Wrn, alone or in combination with Trf2 in mouse embryonic fibroblasts results in increased telomere fusions, which were ablated by Ctip knockdown. We show that WRN regulates alt-NHEJ and shields DSBs from MRE11/CtIP-mediated resection to prevent large deletions and telomere fusions.

POLD1: Central mediator of DNA replication and repair, and implication in cancer and other pathologies.

The evolutionarily conserved human polymerase delta (POLD1) gene encodes the large p125 subunit which provides the essential catalytic activities of polymerase δ (Polδ), mediated by 5'-3' DNA polymerase and 3'-5' exonuclease moieties. POLD1 associates with three smaller subunits (POLD2, POLD3, POLD4), which together with Replication Factor C and Proliferating Nuclear Cell Antigen constitute the polymerase holoenzyme. Polδ function is essential for replication, with a primary role as the replicase for the lagging strand. Polδ also has an important proofreading ability conferred by the exonuclease activity, which is critical for ensuring replicative fidelity, but also serves to repair DNA lesions arising as a result of exposure to mutagens. Polδ has been shown to be important for multiple forms of DNA repair, including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair. A growing number of studies in the past decade have linked germline and sporadic mutations in POLD1 and the other subunits of Polδ with human pathologies. Mutations in Polδ in mice and humans lead to genomic instability, mutator phenotype and tumorigenesis. The advent of genome sequencing techniques has identified damaging mutations in the proofreading domain of POLD1 as the underlying cause of some inherited cancers, and suggested that mutations in POLD1 may influence therapeutic management. In addition, mutations in POLD1 have been identified in the developmental disorders of mandibular hypoplasia, deafness, progeroid features and lipodystrophy and atypical Werner syndrome, while changes in expression or activity of POLD1 have been linked to senescence and aging. Intriguingly, some recent evidence suggests that POLD1 function may also be altered in diabetes. We provide an overview of critical Polδ activities in the context of these pathologic conditions.

Vitamin C alleviates aging defects in a stem cell model for Werner syndrome.

Werner syndrome (WS) is a premature aging disorder that mainly affects tissues derived from mesoderm. We have recently developed a novel human WS model using WRN-deficient human mesenchymal stem cells (MSCs). This model recapitulates many phenotypic features of WS. Based on a screen of a number of chemicals, here we found that Vitamin C exerts most efficient rescue for many features in premature aging as shown in WRN-deficient MSCs, including cell growth arrest, increased reactive oxygen species levels, telomere attrition, excessive secretion of inflammatory factors, as well as disorganization of nuclear lamina and heterochromatin. Moreover, Vitamin C restores in vivo viability of MSCs in a mouse model. RNA sequencing analysis indicates that Vitamin C alters the expression of a series of genes involved in chromatin condensation, cell cycle regulation, DNA replication, and DNA damage repair pathways in WRN-deficient MSCs. Our results identify Vitamin C as a rejuvenating factor for WS MSCs, which holds the potential of being applied as a novel type of treatment of WS.

The effect of RO3201195 and a pyrazolyl ketone P38 MAPK inhibitor library on the proliferation of Werner syndrome cells.

Microwave-assisted synthesis of the pyrazolyl ketone p38 MAPK inhibitor RO3201195 in 7 steps and 15% overall yield, and the comparison of its effect upon the proliferation of Werner Syndrome cells with a library of pyrazolyl ketones, strengthens the evidence that p38 MAPK inhibition plays a critical role in modulating premature cellular senescence in this progeroid syndrome and the reversal of accelerated ageing observed in vitro on treatment with SB203580.

Speeding up the clock: The past, present and future of progeria.

Progeria is a devastating disorder in which patients exhibit signs of premature aging. The most well-known progeroid syndromes include Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner Syndrome (WS). While HGPS and WS are rare, they often result in severe age-associated complications starting in the early developmental period or after the pubertal growth spurt during adolescence, respectively. In addition, patients with HGPS ultimately die of diseases normally seen in the elderly population, with stroke and myocardial infarction as the leading causes of death. Many WS patients develop similar cardiovascular complications but also have an increased predisposition to developing multiple rare malignancies. These premature aging disorders, as well as animal and cell culture models that recapitulate these diseases, have provided insight into the genetics and cellular pathways that underlie these human conditions and have also uncovered possible mechanisms behind normal aging. Here we discuss the history, the types of progeria, and the various pathophysiological mechanisms that drive these diseases. We also address recent medical advances and treatment modalities for patients with progeria.

Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging.

Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2ß. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.