Werner Syndrome Research Articles

Abstract P3-01-20: Werner syndrome protein (WRN) is a negative regulator of cellular migration of breast cancer cells and is associated with the clinical outcome in breast cancer

Abstract Background: Defective human Werner Syndrome protein (WRN), encoded by a single gene is evident in patients with Werner Syndrome (WS) which is an autosomal recessive disorder with appearance of premature aging. Adults with Werner Syndrome present increased incidence of neoplasia, such as thyroid cancers, malignant melanoma, soft tissue sarcomas and some other solid tumours. The present study explored the expression of WRN in breast cancer tissues, its connection with the progression and the survival of breast cancer patients. Impact of WRN on breast cancer cells was also determined. Method: Breast tumours (n=103) together with adjacent background tissues were collected immediately after the surgery and stored at -80°C until use. Total RNA was isolated from the frozen tissues followed by reverse transcription. WRN transcript was determined by Real-Time PCR. Relevance of WRN transcript levels and pathology/clinical features was analysed using SPSS. Knockdown of WRN was carried out with anti-WRN transgenes in human breast cancer cell lines. The In vitro biological effects of altered WRN expression was evaluated by a series of in vitro cellular functional assays. Results: WRN levels in breast tumours were found to be progressively decreased from early to late stage tumours. WRN expression was markedly reduced in tumours from patients who died from breast cancer or with local recurrence, in comparison with those patients who remained disease free. The Kaplan-Meier survival model was used to analyse the overall survival and disease-free survival status of patients. It was found that patients with higher WRN transcript levels had a longer overall survival (95 % CI = 129.6-153.9 months), versus that of patients with lower transcript levels (95 % CI = 112.5-134.9 months). The patients with higher WRN expression tumours also had longer disease-free survival (95 % CI = 119.7-149.1 months) versus that of patients with lower WRN expression tumours (95 % CI = 108.0-131.3 months). Knockdown of WRN in human MDA-MB-231 breast cancer cells markedly increased their migration. Similar effect was also seen in the cells when they were exposed to a small WRN inhibitory compound. WRN were found to be significantly correlated with a group of PUFA metabolising enzymes including α6D (delta-6-desaturate) and 15-LOXB (15-lipooxygenase), indicating that it may be involved in lipid regulated cell motility. Conclusion: There is a correlation between WRN and breast cancer progression and an association between WRN and the survival of patients with breast cancer. Breast cancer cells presented an increase of cell migration following the knockdown of WRN. Collectively, WRN is a putative suppressor of aggressive traits of breast cancer cells which warrants further investigation. Citation Format: Shuo Cai, Lin Ye, Zhe Zhao, Jane Lane, Tracey A. Martin, Eleri Davies, Robert E. Mansel, Wen G. Jiang. Werner syndrome protein (WRN) is a negative regulator of cellular migration of breast cancer cells and is associated with the clinical outcome in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-20.

Checkpoint Defects Elicit a WRNIP1-Mediated Response to Counteract R-Loop-Associated Genomic Instability.

Conflicts between replication and transcription are a common source of genomic instability, a characteristic of almost all human cancers. Aberrant R-loops can cause a block to replication fork progression. A growing number of factors are involved in the resolution of these harmful structures and many perhaps are still unknown. Here, we reveal that the Werner interacting protein 1 (WRNIP1)-mediated response is implicated in counteracting aberrant R-loop accumulation. Using human cellular models with compromised Ataxia-Telangiectasia and Rad3-Related (ATR)-dependent checkpoint activation, we show that WRNIP1 is stabilized in chromatin and is needed for maintaining genome integrity by mediating the Ataxia Telangiectasia Mutated (ATM)-dependent phosphorylation of Checkpoint kinase 1 (CHK1). Furthermore, we demonstrated that loss of Werner Syndrome protein (WRN) or ATR signaling leads to formation of R-loop-dependent parental ssDNA upon mild replication stress, which is covered by Radiorestistance protein 51 (RAD51). We prove that Werner helicase-interacting protein 1 (WRNIP1) chromatin retention is also required to stabilize the association of RAD51 with ssDNA in proximity of R-loops. Therefore, in these pathological contexts, ATM inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Overall, our findings suggest a novel function for WRNIP1 in preventing R-loop-driven genome instability, providing new clues to understand the way replication–transcription conflicts are handled.

Werner综合征一例及其精准诊断

Werner综合征一例及其精准诊断

RECQ DNA Helicases and Osteosarcoma.

The RECQ family of DNA helicases is a conserved group of enzymes that plays an important role in maintaining genomic stability. Humans possess five RECQ helicase genes, and mutations in three of them- BLM, WRN, and RECQL4 - are associated with the genetic disorders Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome (RTS), respectively. These syndromes share overlapping clinical features, and importantly they are all associated with an increased risk of cancer. Patients with RTS have the highest specific risk of developing osteosarcoma compared to all other cancer predisposition syndromes; therefore, RTS serves as a relevant model to study the pathogenesis and molecular genetics of osteosarcoma. The "tumor suppressor" function of the RECQ helicases continues to be an area of active investigation. This chapter will focus primarily on the known cellular functions of RECQL4 and how these may relate to tumorigenesis, as well as ongoing efforts to understand RECQL4's functions in vivo using animal models. Understanding the RECQ pathways will provide insight into avenues for novel cancer therapies in the future.

Candesartan Neuroprotection in Rat Primary Neurons Negatively Correlates with Aging and Senescence: a Transcriptomic Analysis.

Preclinical experiments and clinical trials demonstrated that angiotensin II AT1 receptor overactivity associates with aging and cellular senescence and that AT1 receptor blockers (ARBs) protect from age-related brain disorders. In a primary neuronal culture submitted to glutamate excitotoxicity, gene set enrichment analysis (GSEA) revealed expression of several hundred genes altered by glutamate and normalized by candesartan correlated with changes in expression in Alzheimer's patient's hippocampus. To further establish whether our data correlated with gene expression alterations associated with aging and senescence, we compared our global transcriptional data with additional published datasets, including alterations in gene expression in the neocortex and cerebellum of old mice, human frontal cortex after age of 40, gene alterations in the Werner syndrome, rodent caloric restriction, Ras and oncogene-induced senescence in fibroblasts, and to tissues besides the brain such as the muscle and kidney. The most significant and enriched pathways associated with aging and senescence were positively correlated with alterations in gene expression in glutamate-injured neurons and, conversely, negatively correlated when the injured neurons were treated with candesartan. Our results involve multiple genes and pathways, including CAV1, CCND1, CDKN1A, CHEK1, ICAM1, IL-1B, IL-6, MAPK14, PTGS2, SERPINE1, and TP53, encoding proteins associated with aging and senescence hallmarks, such as inflammation, oxidative stress, cell cycle and mitochondrial function alterations, insulin resistance, genomic instability including telomere shortening and DNA damage, and the senescent-associated secretory phenotype. Our results demonstrate that AT1 receptor blockade ameliorates central mechanisms of aging and senescence. Using ARBs for prevention and treatment of age-related disorders has important translational value.

Functional conservation of RecQ helicase BLM between humans and Drosophila melanogaster

RecQ helicases are a family of proteins involved in maintaining genome integrity with functions in DNA repair, recombination, and replication. The human RecQ helicase family consists of five helicases: BLM, WRN, RECQL, RECQL4, and RECQL5. Inherited mutations in RecQ helicases result in Bloom Syndrome (BLM mutation), Werner Syndrome (WRN mutation), Rothmund-Thomson Syndrome (RECQL4 mutation), and other genetic diseases, including cancer. The RecQ helicase family is evolutionarily conserved, as Drosophila melanogaster have three family members: DmBlm, DmRecQL4, and DmRecQL5 and DmWRNexo, which contains a conserved exonuclease domain. DmBlm has functional similarities to human BLM (hBLM) as mutants demonstrate increased sensitivity to ionizing radiation (IR) and a decrease in DNA double-strand break (DSB) repair. To determine the extent of functional conservation of RecQ helicases, hBLM was expressed in Drosophila using the GAL4 > UASp system to determine if GAL4 > UASp::hBLM can rescue DmBlm mutant sensitivity to IR. hBLM was able to rescue female DmBlm mutant sensitivity to IR, supporting functional conservation. This functional conservation is specific to BLM, as human GAL4 > UASp::RECQL was not able to rescue DmBlm mutant sensitivity to IR. These results demonstrate the conserved role of BLM in maintaining the genome while reinforcing the applicability of using Drosophila as a model system to study Bloom Syndrome.

NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome

Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.

Disease Model of Atherosclerosis Using Werner Syndrome Patient-Derived Induced Pluripotent Stem Cells Reveals Excessive Inflammatory Response Against Ox-LDL

Atherosclerosis is a major cause of various fatal diseases, such as myocardial infarction, stroke, and ischemic heart failure worldwide. The risk factors of atherosclerosis are well known, including hypertension, diabetes mellitus, dyslipidemia, smoking and obesity. Growing evidence also suggests that aging is the strongest determinant of stroke, which is less common before 40 years old (Soler and Ruiz 2010). Werner syndrome (WS) is a rare human inherited disorder characterized by the appearance of premature aging followed by early onset of aging-associated diseases including atherosclerosis. Myocardial infarction and cancer are being the most common causes of death in patients with WS (Oshima et al. 1993). However, despite being one of the most common causes of death, the etiology of atherosclerosis in WS patients have not been well documented yet. Therefore, the aim of the study was to assess in vitro model of atherosclerosis using WS patient-derived induced pluripotent stem cells (iPSC) to better understand the etiology and pathogenesis of atherosclerosis in WS patients. Atherosclerosis is a chronic inflammatory disease in which vascular endothelial cells (VEC), vascular smooth muscle cells (VSMC) and macrophages (Mφ) play interactive roles in the progression of the disease. To obtain these cells in vitro, we induced VEC, VSMC and Mφ from the progenitor cell populations, KDR+/CD34+/TRA-1-60-, KDR+/CD34-/TRA-1-60- and CD43+/CD34+ cells respectively, derived from iPSCs by the iPS-sac method we developed previously (Takayama et al. 2008, 2010). We found by co-culture with C3H10T1/2 mouse mesenchymal stromal cell line, KDR+/CD34+ and KDR+/CD34- cell populations were able to efficiently induce VEC and VSMC respectively on day 17. Especially, VEC induction efficiency was ~ 4 and ~ 21 fold as compared with the previously established method with collagen IV and laminin-411 respectively (Ohta et al. 2016; Sone et al. 2007; Taura et al. 2009). Furthermore, to induce Mφ, iPS-sac-like structures in culture were maintained for two weeks to get CD43+ hematopoietic cells. CD43+ cells were then cultured for an additional 7 days with a cytokine cocktail on C3H10T1/2 feeder layer. After 3 weeks of initiating culture, ~ 54% of CD11b+/CD33+ Mφ-like cells were obtained. To examine the responses of the induced VEC- and Mφ-like cells in atherosclerotic conditions, we applied oxidized low-density lipoprotein (Ox-LDL) at the concentration of 10, 50 and 100 μg/mL, and performed qRT-PCR analysis. It is well known that inflammatory response is triggered with the uptake of LDL both in VEC and Mφ, thereby contribute to local inflammation, cell necrosis, apoptosis, VSMC proliferation and fibrosis. Accordingly, in Ox-LDL treated iPSC-derived VEC and Mφ, mRNA expression of IL-1β, IL-6 and TNF-α were increased in a dose dependent manner. A critical event in the early stages of atherosclerosis is the internalization of lipid particles by VEC, and then, these lipids particles are oxidized to form Ox-LDL in the endothelium, leading to an increase in inflammatory cell adhesion molecules (ICAM-1 and VCAM-1) on the endothelium. Subsequently, monocytes are then recruited and differentiated into Mφ and start to uptake Ox-LDL particles ultimately forming foam-cells (Wu et al. 2017). Next, we were enthusiastic to compare the effect of Ox-LDL for inflammatory response in WS iPSC-derived VEC and Mφ with healthy iPSC-derived VEC and Mφ. Although no differences were observed in the induction efficiency, cell proliferation and mean fluorescent intensity of Ox-LDL uptake between WS- and healthy-iPSC-derived VEC and Mφ, qRT-PCR analysis revealed that, VEC derived from WS-iPSC shows much higher mRNA expression of cell adhesion molecule gene (ICAM-1; 2.5 fold and VCAM-1; 3.6 fold), inflammatory cytokine gene (IL-6; 12.5 fold and TGF-β; 4.3 fold) and endothelial dysfunction related gene (ET-1; 5.9 fold and PAI-1; 25.2 fold) compared with healthy-iPSC-derived VEC. Consequently, our results suggest that the higher risk of atherosclerosis in WS patients might be due to excessive inflammation response against Ox-LDL in WS-iPSC-derived cells, which is independent of other risk factors frequently observed in WS, such as diabetes and hyperlipidemia, and might be implicated to understand the pathogenesis and therapeutic strategy against WS and general atherosclerosis. Disclosures Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Sclerodermalike syndromes: Great imitators

Sclerodermalike syndromes (SLSs) comprise diseases with mucin deposition (eg, scleromyxedema, scleredema), with eosinophilia (eg, eosinophilic fasciitis), metabolic or biochemical abnormalities (eg, nephrogenic systemic fibrosis), or endocrine disorders (eg, POEMS syndrome, or polyneuropathy, organomegaly, endocrinopathy, monoclonal lymphoproliferative disorder, and hypothyroidism). Chronic graft-versus-host disease may also show sclerodermalike skin changes. Inherited progeria syndromes with early aging (eg, Werner syndrome) and a heterogeneous group of hereditary disorders with either skin thickening (eg, stiff skin syndrome) or atrophy and tightening (eg, acrogeria) can also imitate classic systemic sclerosis (SSc). In addition, SLSs can be provoked by several drugs, chemicals, or even physical injury (eg, trauma, vibration stress, radiation). In SLSs, the distribution of skin involvement seems to be atypical compared with SSc. The acral skin involvement is usually missing, and lack of Raynaud phenomenon, scleroderma-specific antinuclear antibodies, the absence of scleroderma capillary pattern, and internal organ manifestations indicate the presence of an SLS. Skin involvement is sometimes nodular, and the underlying tissues can also be affected. For the differential diagnosis, a skin biopsy of the deeper layers including fascia and muscle is required. Histology does not always allow differentiation between SSc and SLSs; therefore, the diagnosis is often based on the distribution, quality of cutaneous involvement, and other accompanying clinical features.

Ligand binding characteristics of the Ku80 von Willebrand domain

The N-terminal von Willebrand domain of Ku80 supports interactions with a Ku binding motif (KBM) that has been identified in at least three other DNA repair proteins: the non-homologous end joining (NHEJ) scaffold APLF, the modulator of retrovirus infection, MRI, and the Werner syndrome protein (WRN). A second, more recently identified Ku binding motif present in XLF and several other proteins (KBMX) has also been reported to interact with this domain. The isolated Ku80 von Willebrand antigen domain (vWA) from Xenopus laevis has a sequence that is 60% identical with the human domain, is readily expressed and has been used to investigate these interactions. Structural characterization of the complexes formed with the KBM motifs in human APLF, MRI, and WRN identify a conserved binding site that is consistent with previously-reported mutational studies. In contrast with the KBM binding site, structural studies indicate that the KBMX site is occluded by a distorted helix. Fluorescence polarization and 19F NMR studies of a fluorinated XLF C-terminal peptide failed to indicate any interaction with the frog vWA. It was hypothesized that availability of this binding site is conditional, i.e., dependent on specific experimental conditions or other repair factors to make the site available for binding. Modulating the fraction of KBMX-accessible binding site mutationally demonstrated that the more open site is capable of binding the KBMXXLF motif peptide. It is suggested that the conditional nature of KBMX binding limits formation of non-productive complexes so that activation-dependent site availability can more optimally support advancing the synapsis process.

P2254Impaired autophagic off-rate causes cardiac aging in progeria mouse model

Abstract Background/Introduction Aging is known to be one of the primary causes of heart failure. Werner syndrome is one of the aging disorder that caused by dysfunction of DNA helicase-regulatory protein (WRN). However, there is little information whether WRN may cause any specific myocardial remodeling and vulnability for heart failure. More interestingly, ample evidences demonstrated DNA damage occurred in progeria causes autophagic disorder, contributing to aging phenotype, in short, autophagy may be a guardian of the genome. Although autophagic disorder has been implicated to cause cardiac remodeling in heart failure; however, it remains uncertain whether autophagic disorder may link to the mechanism of aging-induced cardiac remodeling. Purpose To elucidate whether autophagic disorder may be mechanistically responsible for cardiac aging we hypothesized whether aging-related DNA injury may affect autophagy that may lead to myocardial remodeling. Methods We employed progeria mouse model harboring amino acid (AA) substitution of WRN at position 577 (WRN-K577M), which were evaluated in terms of cardiac function and remodeling at the phase of adult (18 week-old). Results WRN-K577M exhibited diffuse left-ventricular (LV) hypertrophy, enhanced fibrosis, and diastolic LV dysfunction with preserved systolic ejection fraction. DNA microarray analysis of WRN-K577M heart revealed that the 253 genes were upregulated compared to age- and gender-matched wild-type counterpart. Sixteen genes were increased >4 fold higher than wild-type as follows: hypertrophy (Myh7, Klkb11), fibrosis (Fgf21, CTGF), inflammatory molecules (Ap1s3, Pla2g2e, Has1, MMP9), and oxidative stress (catalase). Cardiac aging markers (PARP-1, p53 and γH2AX) increased in heart of WRN-K577M with concomitant increase in oxidative stress (DHE staining) and apoptosis (TUNEL). Notably, autophagic turnover markers (i.e., increased on-rate of autophagy; p62 and LC3-II/I) were increased in myocardium of WRN-K577M, which was refractory to fasting-induced autophagic activation, indicating the on-rate step of autophagy is pathologically augmented under cardiac aging observed in WRN-K577M. In contrast, one of the key regulators of autophagy is the target of rapamycin, TOR kinase, which is the major inhibitory signal that shuts off autophagy with concomitant activation of Akt signaling. In contrast, blockade of the lysosomal fusion into autophagosome by systemic treatment with chloroquine (50 microg/g body weight) reduced LC3-II/I ratio, indicating the retarded off-rate of autophagy mediated by impaired lysosome fusion is presumably responsible for cardiac aging. Conclusion(s) DNA damage impairs autophagy in heart, leading to myocardial oxidative stress. In WRN-mutant progeria model, off-rate disorder of cardiac autophagy is, at least in part, the cause of increase in oxidative stress and inflammation in heart leading to HFpEF.

Congenital muscular dystrophies and congenital myopathies: A study of mutational pattern and phenotypic variability from a tertiary care hospital in India

To characterize the genetic subtypes of Congenital muscular dystrophies (CMD) and Congenital myopathies (CM). A retrospective analysis of CMD and CM (2015–2019) confirmed by exome sequencing. Institutional Ethics Committee approval obtained for the study. CMD group: 15 patients; M:F = 2:3. Onset was congenital in Ullrich CMD(UCMD), FKRP and LAMA2, adult in Bethlem myopathy(BM) and variable in LMNA. Common findings: Contractures, limb girdle and facial weakness, elevated serum creatine kinase. Calf hypertrophy in LAMA2 and FKRP. UCMD (P1,2): Recessive nonsense(p.Gln104ter, p.Gln27ter) mutations in Col6A2 and Col6A3. BM (P3-7): P3-5 had dominant missense mutations in Col6A2(p.Gly564Arg), Col6A1(p.Gly269Ar) and Col12A1(p.Gly982Arg). Co6A3 nonsense(p.Arg59ter) and c.6309+3A>G(5'splice) mutations in P6,7. LAMA2 (P8,9): Compound heterozygous mutations(p.Lys1726ter|p.Arg2604ter; p.Arg2319ter|del 34-37ex): leukoencephalopathy and cerebellar cysts on imaging. LMNA (P10-14): Onset: infancy to 3rd decade. P10-13 had heterozygous missense mutations(p.Arg453Trp, p.Leu102Pro, p.Asn39His and p.Val440Met). Variable phenotypes: P10,11: EDMD, P12: LGMD, P13: RSS with DCM. P14 with biallelic (p.Glu124Argfs*3|p.Glu124Gln) mutations: EDMD with adult progeria features. FKRP (P15): Recessive(p.Pro448Leu) mutation: limb-girdle phenotype. CM group: 12 patients; M:F = 2:1. Infantile onset with motor delay in RYR1, DES and SEPN1. Ptosis was common in all except RYR1. RYR1 (P1-4): P1&2 had opthalmoparesis: compound heterozygous(p.Leu2963Pro| p.Ser783Leu; p.Glu4464Asp|p.Arg628His) mutations. P1 biopsy showed centronuclear myopathy(CNM). P3 had heterozygous(p.Gly3288Arg) mutation. P4 with CM and bullous skin lesions: concomitant RYR1 frameshift(p.Leu108SerfsTer10) and COL7A1 5’splice(c.8620+3G>A) mutations. DES (P5,6): Recessive truncating(p.Arg150Ter, p.Glu320ArgfsTer2) mutations. Variable phenotypes: P5: CM with dilated cardiomyopathy(DCM), P6: Congenital myasthenic syndrome(CMS). SEPN1 (P7,8): Rigid spine syndrome(RSS) with homozygous(p.Met525Arg, p.Ala195Thr) mutations. DNM2 (P9,10): Onset:11-12 years with limb-girdle weakness. Dominant(p.Glu368Lys, p.Val798Met) mutations. This study highlights the mutational pattern and challenging conundrums of genotype-phenotype correlations in CMD and CM.

Investigation of the core binding regions of human Werner syndrome and Fanconi anemia group J helicases on replication protein A

Werner syndrome protein (WRN) and Fanconi anemia group J protein (FANCJ) are human DNA helicases that contribute to genome maintenance. They interact with replication protein A (RPA), and these interactions dramatically enhance the unwinding activities of both helicases. Even though the interplay between these helicases and RPA is particularly important in the chemoresistance pathway of cancer cells, the precise binding regions, interfaces, and properties have not yet been characterized. Here we present systematic NMR analyses and fluorescence polarization anisotropy assays of both helicase-RPA interactions for defining core binding regions and binding affinities. Our results showed that two acidic repeats of human WRN bind to RPA70N and RPA70A. For FANCJ, the acidic-rich sequence in the C-terminal domain is the binding region for RPA70N. Our results suggest that each helicase interaction has unique features, although they both fit an acidic peptide into a basic cleft for RPA binding. Our findings shed light on the protein interactions involved in overcoming the DNA-damaging agents employed in the treatment of cancer and thus potentially provide insight into enhancing the efficacy of cancer therapy.

Evidence for premature aging in a Drosophila model of Werner syndrome.

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by patients' early onset of aging, increased risk of cancer and other age-related pathologies. WS is caused by mutations in WRN, a RecQ helicase that has essential roles responding to DNA damage and preventing genomic instability. While human WRN has both an exonuclease and helicase domain, Drosophila WRNexo has high genetic and functional homology to only the exonuclease domain of WRN. Like WRN-deficient human cells, Drosophila WRNexo null mutants (WRNexoΔ) are sensitive to replication stress, demonstrating mechanistic similarities between these two models. Compared to age-matched wild-type controls, WRNexoΔ flies exhibit increased physiological signs of aging, such as shorter lifespans, higher tumor incidence, muscle degeneration, reduced climbing ability, altered behavior, and reduced locomotor activity. Interestingly, these effects are more pronounced in females suggesting sex-specific differences in the role of WRNexo in aging. This and future mechanistic studies will contribute to our knowledge in linking faulty DNA repair mechanisms with the process of aging.

Abstract 430: Impaired Autophagic Off-rate Causes Cardiac Aging in Progeria Mouse Model

Background/Introduction: Aging is known to be one of the primary causes of heart failure. Werner syndrome is one of the aging disorder due to dysfunction of DNA helicase-regulatory protein (WRN) and exhibiting progressive phenotype of natural aging. Ample evidences demonstrated progeria may promotes autophagic disorder. [PURPOSE] To elucidate whether autophagic disorder may be mechanistically responsible for cardiac aging. Methods: We employed progeria mouse model harboring amino acid (AA) substitution of WRN at position 577 (WRN-K577M, 18 week-old male). Results: WRN-K577M exhibited diffuse left-ventricular (LV) hypertrophy, enhanced fibrosis, and diastolic LV dysfunction with preserved systolic ejection fraction. DNA microarray analysis of WRN-K577M heart revealed that 253 genes were upregulated compared to age- and gender-matched wild-type counterpart. Sixteen genes were increased > 4 fold higher than wild-type as follows: hypertrophy (Myh7, Klkb11), fibrosis (CTGF), inflammatorymolecules (Ap1s3, Pla2g2e, Has1, MMP9), and oxidative stress (catalase). Cardiac aging markers (PARP-1, p53 and γH2AX) increased in heart of WRN-K577M with concomitant increase in oxidative stress (DHE staining) and apoptosis (TUNEL). Notably, autophagic turnover markers (p62 and LC3-II/I) were increased in myocardium of WRN-K577M, which was refractory to fasting-induced increase in on-rate of autophagy. Consistently, one of the key regulators of autophagy is the target of rapamycin, TOR kinase, which is the major inhibitory signal that shuts off autophagy with concomitant activation of Akt signaling. Collectively, these data indicates that the on-rate step of autophagy could be pathologically augmented with disruption of downstream process, i.e. impaired off-rate pathway that modulates turnover of autophagosome and lysosome under cardiac aging observed in WRN-K577M. Changes in autophagosomal and lysosomal accumulation were monitored by specific fluorescence indicators and chloroquine (50 microg/g body weight) augmented lysosomal accumulation with reduced LC3-II/I ratio. Conclusion(s): In WRN-mutant progeria model, off-rate disorder of cardiac autophagy is, at least in part, the cause of cardiac aging.

Reprogramming of Human Peripheral Blood Mononuclear Cell (PBMC) from a patient suffering of a Werner syndrome resulting in iPSC line (REGUi003-A) maintaining a short telomere length

Werner syndrome (WS) is a rare human autosomal recessive disorder characterized by early onset of aging-associated diseases, chromosomal instability, and cancer predisposition, without therapeutic treatment solution. Major clinical symptoms of WS include common age-associated diseases, such as insulin-resistant diabetes mellitus, and atherosclerosis. WRN, the gene responsible for the disease, encodes a RECQL-type DNA helicase with a role in telomere metabolism. We derived a stable iPSC line from 53 years old patient's PBMC, with a normal karyotype, but exhibiting a short telomere length, as a major aspect of the cellular phenotype involved in the pathology.

Epigenetic signatures of Werner syndrome occur early in life and are distinct from normal epigenetic aging processes.

Werner Syndrome (WS) is an adult‐onset segmental progeroid syndrome. Bisulfite pyrosequencing of repetitive DNA families revealed comparable blood DNA methylation levels between classical (18 WRN‐mutant) or atypical WS (3 LMNA‐mutant and 3 POLD1‐mutant) patients and age‐ and sex‐matched controls. WS was not associated with either age‐related accelerated global losses of ALU, LINE1, and α‐satellite DNA methylations or gains of rDNA methylation. Single CpG methylation was analyzed with Infinium MethylationEPIC arrays. In a correspondence analysis, atypical WS samples clustered together with the controls and were clearly separated from classical WS, consistent with distinct epigenetic pathologies. In classical WS, we identified 659 differentially methylated regions (DMRs) comprising 3,656 CpG sites and 613 RefSeq genes. The top DMR was located in the HOXA4 promoter. Additional DMR genes included LMNA, POLD1, and 132 genes which have been reported to be differentially expressed in WRN‐mutant/depleted cells. DMRs were enriched in genes with molecular functions linked to transcription factor activity and sequence‐specific DNA binding to promoters transcribed by RNA polymerase II. We propose that transcriptional misregulation of downstream genes by the absence of WRN protein contributes to the variable premature aging phenotypes of WS. There were no CpG sites showing significant differences in DNA methylation changes with age between WS patients and controls. Genes with both WS‐ and age‐related methylation changes exhibited a constant offset of methylation between WRN‐mutant patients and controls across the entire analyzed age range. WS‐specific epigenetic signatures occur early in life and do not simply reflect an acceleration of normal epigenetic aging processes.

Abstract 2574: Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability

Abstract Werner Syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss of function mutations in WRN lead to premature aging and pre-disposition to cancer. We evaluated synthetic lethality (SL) between not only of WRN, but also of another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN exhibited SL with a DNA mismatch repair protein MutL homolog 1 (MLH1), loss of which is associated with high microsatellite instability (MSI-H). We then demonstrated that MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles and decreased viability in response to WRN knockdown. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors. Citation Format: Lorn Kategaya, Senthil K. Perumal, Jeffrey H. Hager, Lisa D. Belmont. Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2574.

A WRN Nonsense Mutation, p.R1406X, is Not a Risk Factor of Breast Cancer

WRN/RECQL2 plays a crucial role in DNA replication and the maintenance of genome stability. Inactivating mutations in RECQL2 lead to Werner syndrome, a rare autosomal recessive chromosomal disorder characterized by premature aging and associated with genetic instability and increased cancer risk. Whether heterozygous WRN mutations are associated with breast cancer risk has been a matter for debate. In the present study, we identified p.R1406X in the WRN gene in one family case with breast/ovarian cancer using whole exome sequencing. We have investigated the prevalence of this WRN mutation in a total of 828 breast cancer cases, 273 ovarian cancer cases and 445 controls from Bashkortostan Republic (Russia). Our results suggest that the p.R1406X mutation in the WRN gene is not associated with high breast cancer risk.

Abstract 2560: Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells

Abstract Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent large-scale genomic profiling and functional screening of cancer cell lines we identified Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair is frequently detected in human malignancies, in particular in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in the emergence of chromosome breaks, chromatin bridges and micronuclei highlighting defective genome integrity. WRN variants harboring mutations abrogating the ATPase function of WRN helicase fail to rescue the viability phenotype of WRN-depleted MSI-H colorectal cells. Our study suggests that pharmacological inhibition of WRN helicase function might represent a novel opportunity to develop a targeted therapy for MSI-H cancers. Citation Format: Simone Lieb, Silvia Blaha-Ostermann, Elisabeth Kamper, Katharina Ehrenhöfer-Wölfer, Andreas Schlattl, Andreas Wernitznig, Jesse Lipp, Kota Nagasaka, Gerd Bader, Ralph Neumueller, Norbert Kraut, Mark Pearson, Simon Woehrle, Mark Petronczki. Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2560.