WRN inhibition leads to its chromatin-associated degradation via the PIAS4-RNF4-p97/VCP axis.

Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood. Here, we used single-molecule tracking (SMT) in combination with a WRN inhibitor to examine WRN dynamics within the nuclei of living cancer cells. WRN inhibition traps the helicase on chromatin, requiring p97/VCP for extraction and proteasomal degradation in a MSI-H dependent manner. Using a phenotypic screen, we identify the PIAS4-RNF4 axis as the pathway responsible for WRN degradation. Finally, we show that co-inhibition of WRN and SUMOylation has an additive toxic effect in MSI-H cells and confirm the in vivo activity of WRN inhibition using an MSI-H mouse xenograft model. This work elucidates a regulatory mechanism for WRN that may facilitate identification of new therapeutic modalities, and highlights the use of SMT as a tool for drug discovery and mechanism-of-action studies.

KBM-mediated interactions with KU80 promote cellular resistance to DNA replication stress in CHO cells.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

Integrated liver and serum proteomics uncover sexual dimorphism and alteration of several immune response proteins in an aging Werner syndrome mouse model.

Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that males lacking the helicase domain of the Wrn protein orthologue exhibited hepatic transcriptomic and metabolic alterations. In this study, we used a label-free liquid chromatography-tandem mass spectrometry approach to uncover proteins abundance associated with specific biological processes that differed depending on the age (four or ten months) and/or the genotype (wild type or Wrn mutant) in the serum and liver of mice. Principal component analysis of the proteomic data from both serum and hepatic tissue revealed a sexual dimorphism regardless of the age and the genotype of the mice. Moreover, although all Wrn mutant mice exhibited fatty liver by the age of ten months, a significant age and genotype dependent enrichment of proteins involved in lipid and fatty acid metabolic processes were uncovered only in males. Also, a genotype dependent increase in serum oxidant detoxification processes was observed in the serum of Wrn mutant males. Despite these sexual differences, several aspects of the immune system were affected in both females and males. Finally, an increase of specific immunoglobulin molecules was common in the liver and serum of both older Wrn mutant females and males. Such results suggest that specific immunoglobulin variants maybe associated with fatty liver progression in WS.

Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

WRN exonuclease imparts high fidelity on translesion synthesis by Y family DNA polymerases.

Purified translesion synthesis (TLS) DNA polymerases (Pols) replicate through DNA lesions with a low fidelity; however, TLS operates in a predominantly error-free manner in normal human cells. To explain this incongruity, here we determine whether Y family Pols, which play an eminent role in replication through a diversity of DNA lesions, are incorporated into a multiprotein ensemble and whether the intrinsically high error rate of the TLS Pol is ameliorated by the components in the ensemble. To this end, we provide evidence for an indispensable role of Werner syndrome protein (WRN) and WRN-interacting protein 1 (WRNIP1) in Rev1-dependent TLS by Y family Polη, Polι, or Polκ and show that WRN, WRNIP1, and Rev1 assemble together with Y family Pols in response to DNA damage. Importantly, we identify a crucial role of WRN's 3' → 5' exonuclease activity in imparting high fidelity on TLS by Y family Pols in human cells, as the Y family Pols that accomplish TLS in an error-free manner manifest high mutagenicity in the absence of WRN's exonuclease function. Thus, by enforcing high fidelity on TLS Pols, TLS mechanisms have been adapted to safeguard against genome instability and tumorigenesis.

Dietary restriction fails to extend lifespan of Drosophila model of Werner syndrome.

Werner syndrome (WS) is a rare genetic disease in humans, caused by mutations in the WRN gene that encodes a protein containing helicase and exonuclease domains. WS is characterized by symptoms of accelerated aging in multiple tissues and organs, involving increased risk of cancer, heart failure, and metabolic dysfunction. These conditions ultimately lead to the premature mortality of patients with WS. In this study, using the null mutant flies (WRNexoΔ) for the gene WRNexo (CG7670), homologous to the exonuclease domain of WRN in humans, we examined how diets affect the lifespan, stress resistance, and sleep/wake patterns of a Drosophila model of WS. We observed that dietary restriction (DR), one of the most robust nongenetic interventions to extend lifespan in animal models, failed to extend the lifespan of WRNexoΔ mutant flies and even had a detrimental effect in females. Interestingly, the mean lifespan of WRNexoΔ mutant flies was not reduced on a protein-rich diet compared to that of wild-type (WT) flies. Compared to WT control flies, the mutant flies also exhibited altered responses to DR in their resistance to starvation and oxidative stress, as well as changes in sleep/wake patterns. These findings show that the WRN protein is necessary for mediating the effects of DR and suggest that the exonuclease domain of WRN plays an important role in metabolism in addition to its primary role in DNA-repair and genome stability.

A Novel Variant in the WRN Gene Detected in a Case of Early-Onset Severe Insulin Resistance Displaying Some but Not All Hallmarks of Progeroid Werner Syndrome.

OBJECTIVE: Determining the cause of severe insulin resistance and early-onset diabetes in the case of a young woman in which a wide range of differential diagnoses did not apply. RESEARCH DESIGN AND METHODS: Diagnostic workup including medical history, physical examination, specialist consultations, imaging methods, laboratory assessment, and genetic testing carried out by next-generation panel sequencing. RESULTS: After ruling out several differential diagnoses, genetic testing revealed a previously unknown homozygous variant within the canonical splice site of intron 4 in the WRN gene classified as pathogenic. Thus, although not all cardinal clinical criteria according to existing guidelines had been met, the phenotype of our patient was attributed to Werner syndrome (WS), an autosomal-recessive inherited progeroid syndrome. CONCLUSIONS: WS, although rare, must be considered as a differential diagnosis in cases of severe insulin resistance. Moreover, recognized clinical criteria of WS may not lead to diagnosis in all cases.

Sex differences in symptom presentation and their impact on diagnostic accuracy in Werner syndrome.

AIM: Whether sex differences exist in hereditary progeroid syndromes remains unclear. In this study, we investigated sex differences in patients with Werner syndrome (WS), a model of human aging, using patient data at the time of diagnosis. METHODS: The presence of six cardinal signs in the diagnostic criteria was retrospectively evaluated. RESULTS: We found that the percentage of patients with all cardinal signs was higher in males than in females (54.2% vs. 21.2%). By the age of 40 years, 57.1% of male patients with WS presented with all the cardinal signs, whereas none of the female patients developed all of them. In particular, the frequency of having a high-pitched, hoarse voice, a characteristic of WS, was lower in female patients. The positive and negative predictive values for clinical diagnosis were 100% for males and females, indicating the helpfulness of diagnostic criteria regardless of sex. More female patients than male (86.7% vs. 64%) required genetic testing for their diagnosis because their clinical symptoms were insufficient, suggesting the importance of genetic testing for females even if they do not show typical symptoms of WS. Finally, the frequency of abnormal voice was lower in patients with WS harboring the c.3139-1G > C homozygous mutation. CONCLUSION: These results indicate, for the first time, that there are sex differences in the phenotypes of hereditary progeroid syndromes. The analysis of this mechanism in this human model of aging may lead to the elucidation of sex differences in the various symptoms of normal human aging. Geriatr Gerontol Int 2024; 24: 161-167.

Werner helicase mediates the senescence and cell cycle of leukemia cells by regulating DNA repair pathways.

Leukemia is a type of malignant hematological disease that is generally resistant to chemotherapy and has poor therapeutic outcomes. Werner (WRN) DNA helicase, an important member of the RecQ family of helicases, plays an important role in DNA repair and telomere stability maintenance. WRN gene dysfunction leads to premature aging and predisposes humans to various types of cancers. However, the biological function of WRN in cancer remains unknown. In this study, the expression of this RecQ family helicase was investigated in different types of leukemia cells, and the leukemia cell line K562 with high WRN expression was selected to construct a WRN knockdown cell line. The results showed that WRN knockdown inhibited leukemia occurrence and development by regulating the proliferation, cell cycle, differentiation, and aging of cells and other biological processes. The results of transcriptome sequencing revealed that WRN promoted the sensitivity of leukemia cells to the DNA damage inducer Etoposide by regulating cell cycle-related proteins, such as CDC2, cyclin B1, p16, and p21, as well as key proteins in DNA damage repair pathways, such as p53, RAD50, RAD51, and MER11. Our findings show that WRN helicase is a promising potential target for leukemia treatment, providing new ideas for the development of targeted drugs against leukemia.

The identification of a novel mutation (p.I223fs) in WRN associated with Werner syndrome.

PURPOSE: Werner syndrome (WS) is a rare autosomal recessive genetic disease caused by mutations in the WRN gene, and it is characterized by multiple manifestations corresponding to early-onset aging. This study reports the case of a WS patient with a novel WRN mutation. PATIENT AND METHODS: A 36-year-old male patient with WS was evaluated after approval from the local ethics committee. The clinical and biochemical findings of the patient were described. Peripheral blood sample was collected to extract genomic DNA for WRN gene exome sequencing. The three-dimensional (3D) protein structural prediction analysis was performed via the AlphaFold 2.2 program and PyMol software. RESULTS: We report the case of a clinically diagnosed WS patient with consanguineous parents who presented with complex manifestations including early-onset diabetes mellitus, binocular cataracts, cerebral infarction, cerebral atherosclerosis, hypertension, dyslipidemia, hypothyroidism, and suspected meningioma, accompanied by short stature, gray hair, rough skin with subcutaneous fat atrophy, a high-pitched voice, palmoplantar keratoderma, bilateral flat feet, and an indolent deep ulceration on the foot. Exome sequencing identified a novel homozygous frameshift mutation in the WRN gene, c.666-669 del TATT, p.I223fs. The 3D structure prediction showed that premature termination and significant structural changes could occur in the mutant WRN protein. CONCLUSION: We identified a novel homozygous frameshift mutation, p.I223fs, in WRN in a Chinese patient with WS, expanding the spectrum of mutations in WS.

WRN Is a Promising Synthetic Lethal Target for Cancers with Microsatellite Instability (MSI).

Microsatellite instability (MSI), a type of genetic hypermutability arising from impaired DNA mismatch repair (MMR), is observed in approximately 3% of all cancers. Preclinical work has identified the RecQ helicase WRN as a promising synthetic lethal target for patients with MSI cancers. WRN depletion substantially impairs the viability of MSI, but not microsatellite stable (MSS), cells. Experimental evidence suggests that this synthetic lethal phenotype is driven by numerous TA dinucleotide repeats that undergo expansion mutations in the setting of long-standing MMR deficiency. The lengthening of TA repeats increases their propensity to form secondary DNA structures that require WRN to resolve. In the absence of WRN helicase activity, these unresolved DNA secondary structures stall DNA replication forks and induce catastrophic DNA damage.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells supports dual targeting of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

Modified iPOND revealed the role of mutant p53 in promoting helicase function and telomere maintenance.

The G-rich DNA, such as telomere, tends to form G-quadruplex (G4) structure, which slows down the replication fork progression, induces replication stress, and becomes the chromosome fragile sites. Here we described a molecular strategy that cells developed to overcome the DNA replication stress via DNA helicase regulation. The p53N236S (p53S) mutation has been found in the Werner syndrome mouse embryo fibroblast (MEFs) escaped from senescence, could be the driving force for cell escaping senescence. We revealed that the p53S could transcriptionally up-regulate DNA helicases expression, including Wrn, Blm, Timeless, Ddx, Mcm, Gins, Fanc, as well as telomere specific proteins Terf1, Pot1, through which p53S promoted the unwinding of G4 structures, and protected the cells from DNA replication stress induced by G4 stabilizer. By modified iPOND (isolation of proteins on nascent DNA) assay and telomere assay, we demonstrated that the p53S could promote the recruitment of those helicases to the DNA replication forks, facilitated the maintenance of telomere, and prevent the telomere dysfunction induced by G4 stabilizer. Interestingly, we did not observe the function of promoting G4 resolving and facilitating telomere lengthening in the cells with Li-Fraumeni Syndrome mutation-p53R172H (p53H), which suggests that this is the specific gain of function for p53S. Together our data suggest that the p53S could gain the new function of releasing the replication stress via regulating the helicase function and G4 structure, which benefits telomere lengthening. This strategy could be applied to the treatment of diseases caused by telomere replication stress.

A rapid and highly sensitive immunosorbent assay to monitor helicases unwinding diverse nucleic acid structures.

Helicases are crucial enzymes in DNA and RNA metabolism and function by unwinding particular nucleic acid structures. However, most convenient and high-throughput helicase assays are limited to the typical duplex DNA. Herein, we developed an immunosorbent assay to monitor the Werner syndrome (WRN) helicase unwinding a wide range of DNA structures, such as a replication fork, a bubble, Holliday junction, G-quadruplex and hairpin. This assay could sensitively detect the unwinding of DNA structures with detection limits around 0.1 nM, and accurately monitor the substrate-specificity of WRN with a comparatively less time-consuming and high throughput process. Remarkably, we have established that this new assay was compatible in evaluating helicase inhibitors and revealed that the inhibitory effect was substrate-dependent, suggesting that diverse substrate structures other than duplex structures should be considered in discovering new inhibitors. Our study provided a foundational example for using this new assay as a powerful tool to study helicase functions and discover potent inhibitors.

A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection.

Telomeric ends form a loop structure (T-loop) necessary for the repression of ATM kinase activation throughout the normal cell cycle. However, cells undergoing a prolonged mitotic arrest are prone to lose the T-loop, resulting in Aurora B kinase-dependent mitotic telomere deprotection, which was proposed as an anti-tumor mechanism that eliminates precancerous cells from the population. The mechanism of mitotic telomere deprotection has not been elucidated. Here, we show that WRN, a RECQ helicase family member, can suppress mitotic telomere deprotection independently of its exonuclease and helicase activities. Truncation of WRN revealed that N-terminus amino acids 168-333, a region that contains a coiled-coil motif, is sufficient to suppress mitotic telomere deprotection without affecting both mitotic Aurora B-dependent spindle checkpoint and ATM kinase activity. The suppressive activity of the WRN168-333 fragment is diminished in cells partially depleted of TRF2, while WRN is required for complete suppression of mitotic telomere deprotection by TRF2 overexpression. Finally, we found that phosphomimetic but not alanine mutations of putative Aurora B target sites in the WRN168-333 fragment abolished its suppressive effect. Our findings reveal a non-enzymatic function of WRN, which may be regulated by phosphorylation in cells undergoing mitotic arrest. We propose that WRN enhances the protective function of TRF2 to counteract the hypothetical pathway that resolves the mitotic T-loop.

Werner helicase is required for proliferation and DNA damage repair in multiple myeloma.

BACKGROUND: Multiple myeloma (MM), characterized by extensive genomic instability and aberrant DNA damage repair, is a plasma cell malignancy due to the excessive proliferation of monoclonal antibody-producing plasma cells in the bone marrow. Despite the significant improvement in the survival of patients with the development of novel therapeutic agents, MM remains an incurable disease. Werner (WRN) helicase, a member of the RecQ helicase family that contributes to DNA replication, recombination, and repair, has been highlighted in cancer cell survival, yet the role and mechanism of WRN in MM remain unclear. METHODS AND RESULTS: Increased mRNA expression of WRN in newly diagnosed and relapsed CD138+ myeloma plasma cells than normal CD138+ plasma cells and their matched CD138- non-tumorigenic cells were detected by qPCR. Using NSC19630, a specific WRN helicase inhibitor, we further showed decreased cell viability, proliferation, and DNA repair and increased DNA damage and apoptosis in MM cells by MTT assay, cell cycle assay, apoptosis assay, and Western blotting. CONCLUSIONS: The results of the present study demonstrate that WRN is essential in MM cell viability, proliferation, and genomic stability, indicating its inhibition may enhance the efficacy of chemotherapy in MM.

WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures.

The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSß (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.

A homozygous missense variant in the WRN gene segregating in a family with progressive pulmonary failure with recurrent spontaneous pneumothorax and interstitial lung disease.

Interstitial lung disease (ILD) is a condition affecting the lung parenchyma by inflammation and fibrosis and can be caused by various exposures, connective tissue diseases (CTD), and genetic disorders. In this report, a family with five patients having progressive respiratory failure that begins with coughing in adolescence, followed by dyspnea and recurrent spontaneous pneumothorax, and death in early adulthood is presented. The patients were diagnosed to have ILD through clinical and radiological evaluations. Molecular genetic analyses of the family provided two homozygous rare variants in the WRN and SFXN5 genes, co-segregating with the phenotype. The network analyses pointed out that the variant in the WRN, rather than that in the SFXN5 gene, could be the main factor in the existence of the ILD phenotype, putatively through the altered DNA repair and telomere maintenance pathways. In silico analyses suggested that the variant could affect the exonuclease activity or the stability of the WRN protein. Moreover, the adolescent-onset pulmonary phenotype described in the case has not been reported in Werner Syndrome, the only disease known to be associated with biallelic WRN pathogenic variants. Thus, the present phenotype could be either a very atypical presentation of Werner syndrome or a new clinical entity associated with the WRN gene.