WRN Helicase: Is There More to MSI-H than Immunotherapy?

In this issue, Picco and colleagues provide further evidence that WRN inhibitors are synthetically lethal in microsatellite instability-high (MSI-H) cancers and function by blocking the helicase domain of select WRN residues. They demonstrate that WRN inhibitors may be even more effective in a subset of MSI-high tumors with (TA)n repeat expansions, which represents a possible strategy in clinical development. See related article by Picco et al., p. 1457 (1).

Response to Replication Stress and Maintenance of Genome Stability by WRN, the Werner Syndrome Protein.

Werner syndrome (WS) is an autosomal recessive disease caused by loss of function of WRN. WS is a segmental progeroid disease and shows early onset or increased frequency of many characteristics of normal aging. WRN possesses helicase, annealing, strand exchange, and exonuclease activities and acts on a variety of DNA substrates, even complex replication and recombination intermediates. Here, we review the genetics, biochemistry, and probably physiological functions of the WRN protein. Although its precise role is unclear, evidence suggests WRN plays a role in pathways that respond to replication stress and maintain genome stability particularly in telomeric regions.

SNORD88B-mediated WRN nucleolar trafficking drives self-renewal in liver cancer initiating cells and hepatocarcinogenesis.

Whether small nucleolar RNAs (snoRNAs) are involved in the regulation of liver cancer stem cells (CSCs) self-renewal and serve as therapeutic targets remains largely unclear. Here we show that a functional snoRNA (SNORD88B) is robustly expressed in Hepatocellular carcinoma (HCC) tumors and liver CSCs. SNORD88B deficiency abolishes the self-renewal of liver CSCs and hepatocarcinogenesis. Mechanistically, SNORD88B anchors WRN in the nucleolus, promoting XRCC5 interacts with STK4 promoter to suppress its transcription, leading to inactivation of Hippo signaling. Moreover, low expression of STK4 and high expression of XRCC5 are positively correlated with HCC poor prognosis. Additionally, snord88b knockout suppresses mouse liver tumorigenesis. Notably, co-administration of SNORD88B antisense oligonucleotides (ASOs) with MST1 agonist adapalene (ADA) exert synergistic antitumor effects and increase overall murine survival. Our findings delineate that SNORD88B drives self-renewal of liver CSCs and accelerates HCC tumorigenesis via non-canonical mechanism, providing potential targets for liver cancer therapy by eliminating liver CSCs.

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.

Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

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.

Novel WRN Helicase Inhibitors Selectively Target Microsatellite-Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth in vitro and in vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Significance: We report the discovery and characterization of potent, selective WRN helicase inhibitors for MSI cancer treatment, with biomarker analysis and evaluation of efficacy in vivo and in immunotherapy-refractory preclinical models. These findings pave the way to translate WRN inhibition into MSI cancer therapies and provide tools to investigate WRN biology. See related commentary by Wainberg, p. 1369.

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.

Targeting ATP-binding site of WRN Helicase: Identification of novel inhibitors through pocket analysis and Molecular Dynamics-Enhanced virtual screening.

WRN helicase is a critical protein involved in maintaining genomic stability, utilizing ATP hydrolysis to dissolve DNA secondary structures. It has been identified as a promising synthetic lethal target for microsatellite instable (MSI) cancers. However, few WRN helicase inhibitors have been discovered, and their potential binding sites remain unexplored. In this study, we analyzed potential binding sites for WRN inhibitors and focused on the ATP-binding site for screening new inhibitors. Through molecular dynamics-enhanced virtual screening, we identified two compounds, h6 and h15, which effectively inhibited WRN's helicase and ATPase activity in vitro. Importantly, these compounds selectively targeted WRN's ATPase activity, setting them apart from other non-homologous proteins with ATPase activity. In comparison to the homologous protein BLM, h6 exhibits some degree of selectivity towards WRN. We also investigated the binding mode of these compounds to WRN's ATP-binding sites. These findings offer a promising strategy for discovering new WRN inhibitors and present two novel scaffolds, which might be potential for the development of MSI cancer treatment.

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.

Discovering potential WRN inhibitors from natural product database through computational methods.

Microsatellite instability (MSI) is a relatively common feature associated with multiple cancers, and Werner syndrome (WRN) ATP-dependent helicase has been recognized as a novel target for treating MSI cancers, such as colorectal cancer. A small-molecule inhibitor targeting WRN would be a promising strategy for treating colorectal cancer with high MSI expression. In this study, we employed a computer-assisted drug discovery strategy to screen over 30,000 natural product molecules. By using a combination of docking, ligand efficiency, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA), and thermodynamic integration (TI) calculations, we identified MOL008980, MOL010740, MOL011832, T4743, TN1166, and TNP-002173 as potential WRN inhibitors. Subsequent molecular dynamics simulation revealed that these screened natural products possessed better binding dynamic characteristics than ATP substrate and were capable of inhibiting the dynamic process of WRN, making them potential strong ATP competitive inhibitors. In conclusion, our computational approach revealed potential WRN inhibitors from a natural product database, providing a theoretical basis for future research.

Challenges for the Discovery of Non-Covalent WRN Helicase Inhibitors.

The Werner Syndrome RecQ helicase (WRN) is a synthetic lethal target of interest for the treatment of cancers with microsatellite instability (MSI). Different hit finding approaches were initially tested. The identification of WRN inhibitors proved challenging due to a high propensity for artefacts via protein interference, i. e., hits inhibiting WRN enzymatic activities through multiple, unspecific mechanisms. Previously published WRN Helicase inhibitors (ML216, NSC19630 or NSC617145) were characterized in an extensive set of biochemical and biophysical assays and could be ruled out as specific WRN helicase probes. More innovative screening strategies need to be developed for successful drug discovery of non-covalent WRN helicase inhibitors.

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.

Discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for treating cancers with microsatellite instability.

Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.

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.

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.