Targeting the Werner syndrome protein in microsatellite instability cancers: mechanisms and therapeutic potential

Abstract

Microsatellite instability (MSI) is a key feature of cancers with defective DNA mismatch repair, including colorectal, gastric, endometrial, and ovarian cancers. Tumors with MSI depend on the Werner syndrome protein (WRN) for genomic stability, making WRN an attractive therapeutic target. WRN inhibitors exploit the concept of synthetic lethality, inducing selective DNA damage and cell death in MSI tumors while sparing microsatellite stability (MSS) tumor cells or normal cells. Preclinical studies have shown that the efficacy of WRN inhibitors is enhanced when combined with DNA damage response inhibitors or immunotherapy. This review delineates the molecular mechanisms underlying WRN dependency in MSI cancers and explores the therapeutic potential of WRN inhibition. WRN inhibitors represent a promising strategy in precision oncology, especially for MSI tumors, and have the potential to enhance patient outcomes, either as monotherapy or in combination with other treatments.

Introduction

Microsatellite instability (MSI) is a key molecular feature that frequently occurs in solid tumors [1, 2]. Globally, MSI has been identified in over 20 cancer types and is estimated to occur in approximately 3% of all cancers, with hundreds of thousands of new cases diagnosed annually [3]. Among these, colorectal cancer (CRC) with MSI was the first to be recognized, affecting 5–15% of CRC patients [4]. MSI is also present in approximately 22% of cases of gastric cancer [5], 30% of cases of endometrial cancer [6], and 12% of cases of ovarian cancer [7], as well as in other malignancies, such as adrenal cortex cancer, lymphoma, and leukemia [3]. In cancers with MSI, the MMR system is defective. Therefore, it fails to effectively repair these errors, leading to MSI [8]. MMR system defects have various possible causes, including gene mutations and epigenetic modifications. For example, mutations or promoter methylation of MMR genes, such as MLH1, MSH2, MSH6, and PMS2, can lead to the loss or reduction of MMR system function, thereby increasing the risk of MSI [9]. Additionally, certain genetic and environmental factors may affect the function of the MMR system, further promoting the development of cancers with MSI.

Based on its frequency, MSI can be classified into three categories: microsatellite instability-high (MSI-H), microsatellite instability low (MSI-L), and microsatellite stability (MSS). Although conventional therapies, such as chemotherapy, radiotherapy, and immunotherapy, inhibit tumor growth to some extent, they have limited efficacy and associated side effects in a substantial proportion of patients with MSI cancers [10]. Thus, there is an urgent need to explore new therapeutic targets and strategies for the management of cancers with MSI.

In 2019, Chan et al. described the synthetic lethality interaction between Werner syndrome protein (WRN) inhibition and cancers with MSI [11]. The synthetic lethality concept is based on the principle that the simultaneous inactivation of two or more genes leads to irreparable damage and cell death, whereas the loss of a single gene allows cell survival [12, 13]. Loss or inhibition of the WRN results in the accumulation of DNA damage during replication in tumor cells with MSI, ultimately leading to cell death [14]. This mechanism provides a solid rationale for the use of WRN inhibitors as a novel therapeutic strategy for cancers with MSI. A study by Zimmer et al. showed that mutations in the gene encoding the WRN are associated with MSI-H and deficient mismatch repair (dMMR) tumors, suggesting that these tumor cells may be particularly sensitive to WRN inhibitors [4].

The improved understanding of the role of the WRN has driven progress in the development of WRN inhibitors, which have shown promising anti-tumor activity in preclinical models [15]. These inhibitors target WRN helicase activity, preventing effective DNA damage repair in tumor cells and inducing cell death. Baltgalvis et al. and Ferretti et al. demonstrated the significant anti-proliferative and pro-apoptotic effects of WRN inhibitors in tumor cell lines with MSI, especially when used in combination with other DNA damage agents, such as chemotherapeutic drugs or poly-(ADP ribose) polymerase (PARP) inhibitors, resulting in pronounced synergistic effects [16, 17]. This combination strategy not only enhances the therapeutic efficacy of these agents, but it also holds the potential to delay or prevent the development of drug resistance.

This review introduces the WRN as a synthetic lethality target in cancers with MSI feature, followed by a detailed discussion of the mechanisms of action and potential of the WRN as a therapeutic target. We further review the advancements in WRN inhibitor research and assess the therapeutic potential of these agents for the treatment of MSI cancers. Through this review, we aim to provide a deeper understanding of the WRN as a therapeutic target in cancers with MSI and offer insights for future research focused on developing clinically actionable targeted therapies and diagnostics.

WRN helicase: synthetic lethality target in MSI cancers

The WRN is a multifunctional DNA helicase from the RecQ helicase family. In 1996, the defective gene responsible for Werner syndrome was identified and mapped to chromosome 8 through positional cloning. It was subsequently named WRN [18]. Humans possess five RecQ helicases, including RECQL1, BLM, WRN, RECQL4, and RECQL5, all of which are critical for maintaining genomic stability through their roles in DNA replication, repair, and recombination [19, 20]. In addition to WRN deficiency leading to Werner syndrome, mutations in BLM and RECQL4 are linked to Bloom syndrome and Rothmund–Thomson/RAPADILINO/Baller–Gerold syndromes, respectively, which are characterized by genomic instability and cancer susceptibility [21]. While RECQL1 and RECQL5 are not directly associated with specific genetic syndromes, their dysfunction has been implicated in tumorigenesis [19]. The distinct characteristics of these syndromes suggest structural and functional differences among the involved helicases, highlighting the diverse roles of these helicase proteins in maintaining genomic stability.

Structure of WRN

Human RecQ helicases share relatively low domain conservation, including the RecQ-conserved (RQC) domain (encoded by exons 22–25) located downstream of the helicase domain found in the WRN, BLM, RECQL1, and RECQL5, as well as the helicase and RNase D-conserved (HRDC) domain (encoded by exons 30–31) located toward the C-terminus, which is present in the WRN, BLM, and some other members of the RecQ family [22, 23]. WRN is distinguished by its unique enzymatic properties, including a conserved 3′–5′ helicase domain and an unusual 3′–5′ exonuclease domain, setting it apart from other human RecQ helicases [24]. The WRN is the only member of the RecQ family to possess a nuclease exonuclease domain, endowing it with exonuclease activity.

WRN encodes the WRN, which comprises 1432 amino acid residues, with a molecular mass of approximately 160 kDa. Structurally, the WRN can be divided into five major regions from the N-terminus to the C-terminus: the nuclease exonuclease domain, the helicase/ATPase domain, the RQC domain, the HRDC domain, and the nuclear localization signal [22, 25]. The central region of the WRN, encoded by exons 14–21, includes the predicted ATPase and helicase domains, which are essential for its function [18].

The unique structural features of the WRN and its dual helicase and exonuclease activities position it as a crucial player in genomic maintenance. These properties are integral to its function in resolving DNA replication stress, repairing double-strand breaks, and maintaining telomere stability, making the WRN indispensable for cellular homeostasis and an attractive therapeutic target in cancers with MSI. Figure 1 demonstrates the structure of WRN.

Fig. 1.

Domain diagrams of WRN. A Schematic representing the WRN domain structure. The WRN shares four structurally folded domains comprising an ATPase domain, an RQC domain (purple), an exonuclease domain (blue), an HRDC domain (green), and the nuclear localization signal (black). The Zn subdomain (yellow) is located at the C-terminal end of the ATPase domain. B Crystal structure of the catalytic core of WRN helicase with the domains colored individually (Protein Data Bank ID: 6YHR). HRDC, helicase and RNase D-conserved; RQC, RecQ-conserved; WRN, Werner syndrome protein

WRN in DNA repair

WRN is involved in various biological processes; however, its role and mechanisms in DNA damage repair are the most extensively studied. Common types of DNA damage include base modifications caused by oxidation, deamination, and methylation, leading to base substitutions, mismatches, cross-links, and DNA breaks [26]. From a cellular perspective, the WRN is widely distributed in the nucleus, where it interacts with multiple DNA repair proteins to maintain genomic stability and integrity.

In double-strand break repair process, WRN functions through both classical non-homologous end joining (NHEJ) and alternative NHEJ to maintain genomic integrity and prevent MSI tumor cellular death. Classical NHEJ predominantly mediates double-strand break repair throughout the cell cycle and requires several factors, including the heterodimer Ku, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the end-processing factor Artemis, X-ray repair cross-complementing protein 4 (XRCC4)/DNA ligase IV, and various DNA polymerases and end-processing factors [27]. DNA-PKcs interacts with the WRN in a manner that regulates its enzymatic activity, enabling the WRN to form a stable complex with DNA-PK and the DNA-binding subunit Ku at the site of DNA breaks, thereby initiating double-strand break repair [28, 29]. Direct interactions between the Ku70/80 heterodimer and the WRN stimulate the exonuclease activity of the WRN, aiding in the processing of double-strand break ends [30–32]. When Ku70/80 is defective, double-strand break repair switches from classical NHEJ to alternative NHEJ [33]. In alternative NHEJ repair, double-strand break ends are recognized by PARP and MRE11 (double-strand break repair protein), followed by processing of the broken ends by the MRE11/RAD50/NBS1 complex and Ct BP-interacting protein (CtIP) [34]. Notably, when the WRN is recruited to double-strand breaks, it inhibits MRE11 and CtIP recruitment, protecting the broken DNA from 5′ end resection and suppressing alternative NHEJ. The catalytic activity of the WRN is essential for promoting classical NHEJ, whereas non-enzymatic WRN suppresses MRE11/CtIP-mediated alternative NHEJ [35].

WRN in telomere maintenance

MSI has been associated with significant telomere shortening, an early event in tumorigenesis that contributes to genomic instability [36]. In MSI CRC, telomere maintenance mechanisms (TMMs) exhibit subtype-specific activation patterns [37]. Telomeres, which are specialized structures at the ends of chromosomes, are key to safeguarding chromosomal integrity. WRN interacts with components of the shelterin complex, particularly TRF2, to ensure telomere integrity. The shelterin complex is composed of six proteins (TRF1, TRF2, TIN2, Rap1, tripeptidyl peptidase 1 [TPP1], and protein of telomeres 1 [POT1]) that play a crucial role in protecting telomeres from DNA damage surveillance and inappropriate repair [38]. Additionally, the WRN and PARP1 are critical for telomere maintenance and the DNA damage response. The WRN interacts with PARP1, which is a TRF2-associated protein, protects eroded telomeres and modulates TRF2 by poly(ADP-ribosyl)ation, thereby influencing the binding of TRF2 to telomeric DNA [39]. These interactions highlight the multifaceted role of the WRN in telomere stability.

WRN in transcription and RNA metabolism

WRN plays a crucial role in the maintenance of genomic stability during replication stress. In Werner syndrome cells lacking the WRN, impaired ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR)-checkpoint kinase 1 (CHK1) signaling leads to the accumulation of R-loops and genomic instability, while ataxia-telangiectasia mutated (ATM) pathway activation can partially alleviate this issue [40]. The WRN is involved in the ATR-mediated replication checkpoint response, with ATR and ATM maintaining replication fork integrity by regulating WRN function [41]. Following DNA damage, the RNA helicase Ddx19 migrates from the nuclear pore to the nucleus in an ATR/CHK1-dependent manner to eliminate R-loops generated by replication–transcription conflicts, thereby preventing DNA damage and genomic instability [42]. R-loops are DNA–RNA hybrid structures, and their imbalance can lead to replication stress and genomic instability [43]. In cells deficient in the WRN, the ATR/CHK1 pathway is compromised, resulting in disrupted nuclear localization of Ddx19 and subsequent R-loop clearance defects. These findings highlight the intricate relationship between the WRN, the ATR/CHK1 pathway, and R-loop resolution in maintaining genomic stability during replication stress.

Tumorigenesis-induced replication stress triggers transcription–replication conflicts, which are associated with genomic instability. A previous study showed that the WRN plays a vital role in transcription mediated by RNA polymerase I (Pol I) and RNA polymerase II (Pol II). As part of the RNA Pol I-associated complex, the WRN accelerates ribosomal RNA (rRNA) transcription, and cells deficient in the WRN exhibit decreased rRNA transcription [44]. Similarly, the WRN is involved in the transcription of RNA Pol II, with transcription efficiency reduced in Werner syndrome cells compared with healthy cells [45]. The WRN is also a critical mediator of the stimulation of RNA Pol I activity by growth factors, and it plays a role in promoter clearance during Pol I transcription [46]. Furthermore, the WRN affects the export of messenger RNA (mRNA) from the nucleus through its interaction with nuclear RNA Export Factor 1, influencing cellular protein homeostasis and overall protein synthesis [47]. Collectively, these findings underscore the significant impact of the WRN on cellular transcription and mRNA processing.

WRN in aging and autophagy

WRN is intricately linked to aging and autophagy, and defects in WRN or mutations in its encoding gene are associated with several genetic disorders. Notably, mutations in WRN cause Werner syndrome, a rare autosomal recessive disorder characterized by early onset symptoms, such as cataracts, osteoporosis, diabetes mellitus, and atherosclerosis [48]. The International Registry of Werner Syndrome and the Japanese Werner Consortium have identified numerous WRN mutations, including genomic rearrangements, intronic mutations, and ethnicity-specific alterations [49, 50]. These mutations impair the function of the WRN, driving aging-related cellular and organismal phenotypes [51]. Symptom onset can be attributed to compromised WRN function, leading to decreased DNA repair and impaired telomere maintenance, in turn triggering cellular senescence and genomic instability.

Cellular nicotinamide adenine dinucleotide (NAD +) depletion is a major driver of metabolic dysfunction in patients with Werner syndrome, resulting in dysregulation of mitochondrial homeostasis [52]. NAD⁺ plays a crucial role in aging and neurodegeneration. WRN regulates NMNAT1, a key enzyme involved in NAD⁺ biosynthesis. Supplementation of NAD⁺ precursors, such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), has been shown to restore the metabolic profile in models of Werner syndrome [53]. This restoration occurs through the improvement in mitochondrial quality via mitophagy, a process mediated by UNC-51-like kinase 1, an upstream regulator of autophagy. Consequently, these interventions have demonstrated the potential to extend the lifespan of the Werner syndrome model. These findings underscore the vital role of WRN in mitigating aging-related cellular dysfunction and cellular autophagy, offering insights into potential therapeutic strategies for aging associated disorders.

In summary, WRN plays multiple essential roles in biological processes, including DNA repair and maintenance of telomere stability. The proper execution of these roles is crucial for maintaining normal physiological functions in cells, preventing cellular transformation, and extending the cellular lifespan. Figure 2 demonstrates the functions of WRN.

Fig. 2.

Functions of WRN. Mutations in WRN cause Werner syndrome, and patients with Werner syndrome often present with symptoms such as cataracts, osteoporosis, diabetes mellitus, and atherosclerosis. The WRN is involved in DNA repair, telomere maintenance, aging and autophagy, and replication stress, among other pathways. WRN, Werner syndrome protein; c-NHEJ, classical non-homologous end joining; alt-NHEJ, alternative NHEJ repair. Created with BioRender.com

Roles of WRN inhibitors in the treatment of MSI cancers

WRN inhibitors have emerged as a promising therapeutic drug class, garnering increasing attention for their therapeutic potential in MSI cancers. Several WRN inhibitors have been developed and have demonstrated promising anti-tumor effects in preclinical studies [16, 17]. These inhibitors work through binding to specific sites on the WRN and interfering with its enzymatic activities, thereby effectively inhibiting the growth and spread of tumors cells with MSI. Certain WRN inhibitors selectively bind to the C727 site on the WRN, leading to loss of its helicase activity, in turn triggering the apoptosis of tumors cells with MSI [16]. Combining WRN inhibitors with immune checkpoint inhibitors further enhances the anti-tumor efficacy of these agents, offering new therapeutic options for cancers with MSI [54].

Anti-tumor mechanisms of WRN inhibitors: synthetic lethality

The induction of synthetic lethality by inhibiting the function of WRN in MSI cancer cells. Defects in the DNA MMR system prone to form non-B DNA secondary structures [14] and activate ATR-dependent WRN phosphorylation. Phosphorylated WRN facilitates to unwind the secondary structures and allow to proceed replicate [55, 56]. In the absence of the WRN, the structure-specific Mus81-Eme1 endonuclease, along with its scaffold protein SLX4, attempt to cleave the secondary structures at numerous thymine–adenine (TA) repeat and restore the replication fork functionality [14]. However, excessive Mus81-mediated cleavage leads to DNA end resection, chromosomal fragmentation, and ultimately cell death [57]. Recent studies have revealed that the growth of cancers with MSI is highly dependent on the WRN helicase. Lieb et al. used RNA interference to silence WRN expression in multiple MSI and MSS cell lines and subsequently evaluated apoptosis [58]. Their findings demonstrated that WRN silencing induced significant apoptosis of MSI-H cells, while no apoptotic effects were observed on MSS cells. Therefore, the synthetic lethality observed in MMR-deficient cells is independent of the exonuclease activity of the WRN and specifically relies on its helicase function [11].

WRN inhibitors function by disrupting the helicase activity of the WRN, thereby preventing the resolution of non-canonical secondary DNA structures, leading to replication fork stalling and DNA damage [59]. This damage activates DNA damage response pathways, including promoting the activation of ATM and CHK2, resulting in cell cycle arrest and apoptosis [60]. Additionally, WRN inhibitors may induce cell death through other mechanisms, such as compromising telomere stability and interfering with DNA repair. Studies have shown that WRN inhibitors exhibit significant anti-tumor activity in cancers with MSI. For example, in colorectal, gastric, and endometrial cancers with MSI-H, WRN inhibitors selectively kill tumor cells with minimal impact on healthy cells [11]. This selective cytotoxicity underscores the potential of WRN inhibitors as promising anti-cancer agents (Fig. 3).

Fig. 3.

Mechanism of WRN inhibitors in the treatment of MSI cancers. TA-dinucleotide repeats exhibit high instability and undergo extensive expansions in cells with MSI. This feature makes cells with MSI particularly vulnerable to disruption of genomic maintenance mechanisms. The preferential dependency of cells with MSI on the WRN arises from their inherent deficiencies in MMR, which results in an inability to effectively manage replication stress and repair DNA damage. WRN inhibition exacerbates these vulnerabilities, leading to the accumulation of DNA errors, replication fork collapse, and eventual cell death in cells with MSI. Conversely, cells with MSS, which possess functional MMR pathways, are less reliant on the WRN for the maintenance of genomic integrity, allowing them to survive WRN inhibition. WRN inhibition selectively impairs the viability of cells with MSI while sparing cells with MSS, highlighting the WRN as a promising synthetic lethality target. MMR, mismatch repair; MSI, microsatellite instability; MSS, microsatellite stability; WRN, Werner syndrome protein. Created with BioRender.com

Research status of WRN inhibitors

Researchers have adopted various strategies to develop effective WRN inhibitors, with chemoproteomics platforms serving as a pivotal tool. These platforms allow researchers to quantify the covalent binding of small molecules to amino acid residues within intact cells or cell lysates, enabling the identification of compounds with potential inhibitory activity. Additionally, target-based mass spectrometry-driven chemical proteomics approaches have been used to assess the selectivity and potency of compounds that inhibit the WRN. These methods provide valuable insights into the molecular interactions and mechanisms underlying WRN inhibition, accelerating the discovery of targeted therapeutic agents.

Several WRN inhibitors have entered the development pipeline, demonstrating significant progress. Among them, HRO761 and VVD-133214 stand out as representative WRN inhibitors, exhibiting promising anti-tumor activity in preclinical studies [16, 17]. HRO761 is a non-covalent allosteric WRN inhibitor that was developed by Novartis [17]. It binds allosterically at the interface of the D1 and D2 helicase domains, locking the WRN in an inactive conformation. Preclinical studies have demonstrated the efficacy of HRO761 in both cell-derived xenograft and patient-derived xenograft (PDX) models, showing promising therapeutic potential against cancers with MSI-H. In vivo studies have indicated that combining HRO761 with the topoisomerase I (TOP1) inhibitor irinotecan enhances DNA damage and antiproliferative effects, leading to complete tumor regression. Ongoing phase I clinical trials aim to evaluate the safety, tolerability, and optimal dosage of HRO761 as a monotherapy or in combination with other agents, such as pembrolizumab or irinotecan, with the goal of optimizing treatment strategies. VVD-133214 (also known as RO7589831) is another noteworthy WRN inhibitor [16]. VVD-133214 acts covalently and allosterically on WRN, which selectively binding to the C727 site to inhibit its helicase activity. This compound induces widespread DNA double-strand breaks, nuclear swelling, and cell death in cancer cells with MSI-H. VVD-133214 has demonstrated robust anti-tumor activity in several PDX models of CRC with MSI-H.

HRO761 and VVD-133214 have progressed to phase I clinical trials, while several other WRN inhibitors are in the preclinical stage of research. For instance, SL-4, which was co-developed by GlaxoSmithKline and IDEAYA Biosciences, and ISM2196, an artificial intelligence-driven discovery by Insilico Medicine, have demonstrated anti-tumor efficacy in preclinical studies. Additionally, a novel class of thiophen-2-ylmethylene bis-dimedone derivatives has been identified. These agents show potential therapeutic activity when used to inhibit the WRN [61]. Furthermore, computational drug discovery strategies have been implemented to identify potential WRN inhibitors from natural products, such as MOL008980 [62]. Figure 4 shows the structure of two WRN inhibitors, HRO761 and VVD-133214. Additionally, several WRN inhibitors and their research information are given in Table 1.

Fig. 4.

Structures of WRN inhibitors. A, C Chemical structures. B, D X-ray structures. B HRO-761 bound to the WRN (Protein Data Bank ID: 8PFO). D VVD-133214 bound to the WRN (Protein Data Bank ID: 7GQU). Inhibitors are labeled in yellow, WRN residues in the binding pocket are labeled in white, and hydrogen bonds are represented by green dashes. WRN, Werner syndrome protein

Table 1.

The WRN inhibitors

Drug	Cancer type	Phase	Enrollment	Trial identifier
HRO-761	dMMR*/MSI-H* colorectal cancer MSI-H solid tumors dMMR solid tumors Turcot syndrome	1	327	NCT05838768
RO-7589831/VVD-133214	Advanced malignant solid neoplasm MSI-H cancer dMMR solid tumors	1	220	NCT06004245
GH-1581	Cancer	Preclinical research	N/A	N/A
ISM-9342A	MSI-H cancer	Preclinical research	N/A	N/A
SL-4	Gastrointestinal neoplasms	Preclinical research	N/A	N/A
ISM-2196	Cancer	Preclinical research	N/A	N/A
CC-003	Solid tumors	Preclinical research	N/A	N/A
MOMA-341	Colorectal cancer Endometrial carcinoma Stomach cancer	Preclinical research	N/A	N/A
WRN* inhibitors (Sun Yat-Sen University)/Compound h6	Cancer	Preclinical research	N/A	N/A
SP-010	Cancer	Preclinical research	N/A	N/A

*dMMR, deficient mismatch repair; MSI-H, microsatellite instability-high; WRN, Werner syndrome protein

Despite these advances, the complexity of cancers with MSI and their relationship to tumor evolution present significant challenges for clinical application [63]. Successful translation of WRN inhibitors into clinical practice requires them to meet certain conditions, and issues such as selecting the appropriate patient population remain to be further explored. WRN inhibitors offer a new treatment option for patients with MSI-H cancers, particularly those who are resistant to standard therapies and immunotherapies. With continued research and technological advancements, it is anticipated that more efficient and less toxic WRN inhibitors will be discovered, facilitating new breakthroughs and progress in the field of cancer treatment. Additionally, the development of WRN inhibitors will further our understanding of WRN function and its relationship with diseases, providing strong support for precision medicine and personalized therapy.

Functions of WRN and WRN inhibitors in various MSI cancers

WRN plays an important role in CRC, and its aberrant expression is closely associated with CRC onset and progression. Several studies have found that epigenetic inactivation of WRN, through promoter hypermethylation, is commonly present in various tumors. This leads to the loss of WRN-related exonuclease activity, increased chromosomal instability, and heightened sensitivity to chemotherapeutic agents [64, 65]. In CRC, high methylation of WRN is a marker for predicting a good clinical response to the TOP1 inhibitor irinotecan [64], which is used in the treatment of this type of cancer. WRN mutations and deletions have also been linked to CRC development, particularly in right-sided CRC, CRC with MSI, or CRC with dMMR [4, 58]. In a study of a 32-year-old male with early onset familial CRC [66], a pathogenic frameshift mutation in WRN (p.R389Efs*3) was identified, which was associated with genetic susceptibility to CRC. MSI analysis of the patient revealed the MSI phenotype, and immunohistochemistry staining revealed the absence of MSH2 and MSH6 protein expression. This study suggests that loss of WRN function may contribute to the development of CRC and warrants further investigation in the context of hereditary cancer diagnosis.

A study by Picco et al. showed that WRN dependence was a common feature of synthetic lethality in clinical preclinical models of 60 heterogeneous cases of CRC with dMMR [67]. Furthermore, WRN inhibitors may be more effective in some MSI-H tumors with (TA)n repeat expansions [54]. Hao et al. demonstrated that WRN deficiency strongly induced p53 and its downstream apoptotic target p54 upregulated modulator of apoptosis (PUMA) in CRC cells with MSI [68]. Inhibiting the growth of CRC with MSI by WRN deficiency or treatment with the RecQ helicase inhibitor ML216 was found to be p53/PUMA-dependent. Their research supports the WRN as a promising therapeutic target in p53 wild-type CRC with MSI. However, Lieb et al. and Zong et al. observed that p53-deficient HCT116 cells showed similar sensitivity to small interfering/short hairpin RNA targeting the WRN to p53 wild-type cells [58, 69]. Certain factors, such as the use of different tissues to produce MSI cell models, overexpression of certain cytokines, or gain-of-function mutations, may influence the observed effects of WRN inhibitors.

Endometrial cancer is another type of cancer with a high frequency of MSI, particularly in patients with Lynch syndrome. Similar to CRC, endometrial cancer cells with MSI rely on the WRN to maintain their genomic stability. One study reported a 51-year-old female patient with synchronous endometrial and ovarian cancer who exhibited a pathogenic mutation in WRN, [c.4109del, p.(Asn1370ThrfsTer23)] [70]. Both tumors displayed endometrial-like histological features and showed loss of MutL protein homolog 1 (MLH1) and PMS protein expression. This cases contributed to a deeper understanding of the etiology of Lynch-like syndrome, emphasizing the necessity of specific genetic testing and genetic counseling in hereditary cancer syndromes.

In summary, WRN and its encoded protein, the WRN, play crucial roles in various types of cancer, with abnormal WRN expression closely associated with cancer development and progression. These findings suggest that WRN inhibitors could become an important tool in the treatment of cancers with MSI, offering a new option for precision medicine in clinical practice.

Discussion

MSI is an important aspect of cancer pathogenesis, particularly in certain types of cancer. Compared with patients with MSS cancers, clinical studies have shown that patients with MSI cancers exhibit better immune responses, an enhanced ability to suppress tumor growth, and a better prognosis after surgical treatment [71]. However, patients with metastatic cancers with MSI often have a poor prognosis, with many developing resistance to chemotherapy and exhibiting a poor response to immunotherapy. This population of patients requires more effective therapeutic options [72]. The potential of WRN as a therapeutic target in cancers with MSI is increasingly recognized. WRN inhibition has significant effects on tumors with MSI, while it has minimal impact on tumors with MSS. The synthetic lethality observed in cancers with MSI upon targeting the WRN provides a foundation for developing new therapeutic strategies that selectively target cancer cells while sparing healthy cells.

Preclinical studies have shown that WRN inhibitors exhibit significant anti-tumor activity across various models of cancer with MSI. These studies not only underscore the direct effects of WRN inhibition in MSI cancer therapy, but also suggest that WRN inhibitors may work synergistically with other treatments, such as immunotherapy and chemotherapy. For instance, tumor cells with MSI may accumulate more DNA damage and mutations by inhibiting WRN, leading to increased neoantigen formation. Given that MSI tumors typically respond well to immunotherapy, combining WRN inhibitors with immune-based therapies could result in stronger anti-tumor effects. However, several challenges must be addressed before WRN inhibitors be effectively applied in clinical treatments. A study has reported that WRN performed essential physiological functions in healthy cells, raising concerns about potential damage to healthy tissues during tumor targeting. It indicated that achieving over 95% WRN inhibition was necessary to elicit substantial cytotoxic effects, as even minimal WRN activity sustained the viability of cells with MSI [69]. Additionally, systemic WRN inhibition might induce complications resembling Werner syndrome [51], so the cumulative effects of WRN inhibitor dosing warrant careful consideration. Although promising results have been demonstrated in preclinical studies, while the safety, efficacy, and optimal dosing of WRN inhibitors in humans remain to be validated through rigorous clinical trials. Furthermore, the heterogeneity of tumors with MSI could influence the therapeutic outcomes of WRN inhibitors. To address these challenges, individualized treatment strategies tailored to specific patient profiles are essential. A detailed analysis of the function of the WRN in various tumor subtypes is needed to explore the differential therapeutic effects and mechanisms of WRN inhibitors in distinct types of cancer with MSI. Such efforts will be critical to maximizing the therapeutic potential of WRN inhibitors and overcoming barriers to their clinical application.

Despite these challenges, targeting the WRN in cancers with MSI remains a highly promising therapeutic strategy. In vitro studies have demonstrated that WRN inhibitors sensitize cancer cells to DNA-damaging agents and modulate the tumor microenvironment to enhance immune recognition. Moreover, silencing the WRN increases the chemotherapeutic efficacy of camptothecin in cancer cells by impairing DNA damage repair [73]. In models of CRC with MSI-H that are resistant to targeted therapies, chemotherapy, or immunotherapy, WRN dependency persists, supporting the therapeutic potential of WRN inhibitors as a monotherapy or for use in combination with other treatments [67]. Zong et al. found that partial WRN degradation combined with low doses of ATR inhibitors significantly enhanced their combined efficacy, achieving near-complete WRN inactivation [69]. The result highlights the potential for synergistic application of WRN and ATR inhibitors in the treatment of cancers with MSI. These findings position WRN inhibitors as a novel therapeutic approach for such cancers, particularly when used in combination with existing therapies, such as immunotherapy, chemotherapy, and radiotherapy, to enhance treatment outcomes.

The clinical translation of WRN inhibitors requires the existing limitations to be overcome, which calls for collaborative efforts across the scientific community. Future research should focus on developing more effective and specific WRN inhibitors while minimizing potential side effects. Determining the minimal residual WRN levels required to sustain the survival of cancer with MSI-H represents a critical avenue of future research to design next-generation WRN inhibitors. Additionally, identifying biomarkers that are predictive of WRN inhibitor efficacy is essential for stratifying patients who are most likely to benefit from this therapeutic strategy. The complexity and diverse molecular characteristics of cancers with MSI underscore the need for a deeper understanding of how WRN inhibition influences tumor biology and patient outcomes. Exploring the roles of the WRN in different cancer subtypes with MSI could guide the development of more targeted and effective therapies. Furthermore, addressing potential resistance to WRN inhibitors is crucial. Therefore, future studies should investigate combination strategies with other drugs or treatments to mitigate resistance and enhance efficacy.

Conclusion

The WRN is a multifunctional DNA helicase that is involved in various biological processes, including DNA damage repair, telomere maintenance, autophagy, and genomic stability. Cancers with MSI are primarily driven by defects in the MMR system. WRN dependency can be leveraged as a synthetic lethality strategy for cancers with MSI. Several WRN inhibitors targeting MSI cancers have been developed and are in various stages of research. Compounds such as HRO761 and VVD-133214 have been shown to effectively inhibit the proliferation of tumor cells with MSI with low toxicity. Moreover, combining WRN inhibitors with existing therapies has shown promising synergistic effects, offering new therapeutic strategies for cancers with MSI. As our understanding of WRN biology and MSI tumor pathophysiology deepens, and as more clinical trial data are generated, WRN-based synthetic lethality strategies are expected to become a cornerstone in the treatment of cancers with MSI.

Author contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. Writing—original draft preparation was done by Shuling Chen, Zhiming Wang; visualization was done by Shuling Chen; writing—review and editing was done by Mengmeng Xu; funding acquisition was done by Yongsheng Zhang, Zhifei Cao, Mengmeng Xu; and supervision was done by Yongsheng Zhang.

Funding

This work was supported by National Natural Science Foundation of China (12275192), the Science and Technology Plan Project of Suzhou (SKY2022150), and the project from State Key Laboratory of Radiation Medicine and Protection (GZK1202308, GZK12024046).

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Conflict of interest

The authors declare no conflict of interest.