PROTAC介导的WRN解旋酶条件降解作为选择性杀伤具有微卫星不稳定性的癌症细胞的潜在策略-动脉网

PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability

Nature 等信源发布 2024-09-06 19:40

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AbstractMultiple studies have demonstrated that cancer cells with microsatellite instability (MSI) are intolerant to loss of the Werner syndrome helicase (WRN), whereas microsatellite-stable (MSS) cancer cells are not. Therefore, WRN represents a promising new synthetic lethal target for developing drugs to treat cancers with MSI.

摘要多项研究表明，具有微卫星不稳定性（MSI）的癌细胞不耐受Werner综合征解旋酶（WRN）的丧失，而微卫星稳定（MSS）癌细胞则不耐受。因此，WRN代表了一种有前途的新的合成致死靶标，用于开发用MSI治疗癌症的药物。

Given the uncertainty of how effective inhibitors of WRN activity will prove in clinical trials, and the likelihood of tumours developing resistance to WRN inhibitors, alternative strategies for impeding WRN function are needed. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that target specific proteins for degradation.

鉴于WRN活性抑制剂在临床试验中如何有效证明的不确定性，以及肿瘤对WRN抑制剂产生耐药性的可能性，需要替代策略来阻止WRN功能。蛋白水解靶向嵌合体（PROTAC）是靶向特定蛋白质降解的异双功能小分子。

Here, we engineered the WRN locus so that the gene product is fused to a bromodomain (Bd)-tag, enabling conditional WRN degradation with the AGB-1 PROTAC specific for the Bd-tag. Our data revealed that WRN degradation is highly toxic in MSI but not MSS cell lines. In MSI cells, WRN degradation caused G2/M arrest, chromosome breakage and ATM kinase activation.

在这里，我们设计了WRN基因座，以便将基因产物与溴结构域（Bd）标签融合，从而能够使用对Bd标签特异的AGB-1 PROTAC进行条件性WRN降解。。在MSI细胞中，WRN降解导致G2/M停滞，染色体断裂和ATM激酶激活。

We also describe a multi-colour cell-based platform for facile testing of selective toxicity in MSI versus MSS cell lines. Together, our data show that a degrader approach is a potentially powerful way of targeting WRN in MSI cancers and paves the way for the development of WRN-specific PROTAC compounds..

我们还描述了一个基于多色细胞的平台，用于轻松测试MSI与MSS细胞系的选择性毒性。总之，我们的数据表明，降解剂方法是在MSI癌症中靶向WRN的潜在有效方法，并为WRN特异性PROTAC化合物的开发铺平了道路。。

IntroductionTo prevent the potentially genome-destabilizing effects of DNA damage, cells are equipped with a range of DNA damage repair (DDR) pathways. The DNA mismatch repair (MMR) pathway, for example, detects and repairs mismatches introduced during genome replication1,2. Efficient MMR requires the products of several key genes including MLH1, PMS2, MSH2 and MSH63.

。例如，DNA错配修复（MMR）途径检测和修复基因组复制过程中引入的错配1,2。有效的MMR需要几个关键基因的产物，包括MLH1，PMS2，MSH2和MSH63。

MSH2/6 dimers bind to mismatched base errors, while MLH1-PMS2 direct the excision step and fill-in synthesis4,5. Germline mutations in MMR genes cause diseases such as Lynch syndrome, which is associated with a predisposition to multiple cancers including colon and endometrial cancers6,7. In regions of the genome containing microsatellites—short tracts of repetitive sequences, inappropriate primer-template annealing and/or polymerase slippage can lead to insertion/deletions loops (IDLs) which can be repaired by MMR8,9.

MSH2/6二聚体与不匹配的碱基错误结合，而MLH1-PMS2指导切除步骤并填充合成4,5。MMR基因的种系突变会导致Lynch综合征等疾病，这与多种癌症的易感性有关，包括结肠癌和子宫内膜癌6,7。在含有微卫星短片段重复序列的基因组区域中，不适当的引物-模板退火和/或聚合酶滑移可导致插入/缺失环（IDL），可通过MMR8,9修复。

Microsatellites become unstable in MMR-defective cells, in the form of expansions that can be detected in PCR-based assays10. Microsatellite instability (MSI) has been observed in approximately 15% of all colorectal cancers, and in endometrial, ovarian and gastric cancers11,12. It has also been estimated that 22% of all western gastric cancers are MSI + .

微卫星在MMR缺陷细胞中变得不稳定，其形式是可以在基于PCR的测定中检测到的扩增10。。据估计，所有西方胃癌中有22%是MSI+。

While it is not well understood whether genetic or environmental factors drive this phenomenon, gastric cancers occur at particularly high frequencies in South Korea (42 per 100K age-standardized rate), Mongolia (32 per 100K age-standardized rate), Japan (30 per 100K age-standardized rate), and China (23 per 100K age-standardized rate)13,14.There are currently no therapies for MSI cancers that directly target deficiencies in the MMR system15,16.

虽然目前尚不清楚遗传或环境因素是否会导致这种现象，但在韩国（每100K年龄标准化率为42），蒙古（每100K年龄标准化率为32），日本（每100K年龄标准化率为30）和中国（每100K年龄标准化率为23）13,14，胃癌的发生频率特别高。目前还没有针对MMR系统缺陷的MSI癌症治疗方法15,16。

There is evidence however, that MSI tumours are amenable to immune checkpoint blockade17,18,19,2.

然而，有证据表明，MSI肿瘤可以接受免疫检查点封锁17,18,19,2。

Data availability

数据可用性

All data supporting the findings of this study are available from the corresponding author upon reasonable request.

所有支持这项研究结果的数据都可以在合理的要求下从通讯作者那里获得。

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Download referencesAcknowledgementsWe thank the excellent technical support of the MRC PPU including the DNA Sequencing Service, Tissue Culture Team, and the Reagents and Services Team. We thank Arlene Rennie and Rosemary Clarke (Centre for Advanced Scientific Technologies, School of Life Sciences Dundee) for extensive help with FACS-based cell sorting and flow cytometry experiments.

下载参考文献致谢我们感谢MRC PPU的出色技术支持，包括DNA测序服务，组织培养团队以及试剂和服务团队。我们感谢Arlene Rennie和Rosemary Clarke（邓迪生命科学学院高级科学技术中心）在基于FACS的细胞分选和流式细胞仪实验方面的广泛帮助。

We thank Gillian Smith (Jacqui Wood Cancer Centre, Ninewells Hospital Dundee) for kindly providing Caov-3 ovarian cancer cells, and Daniel Durocher (Lunenfeld-Tannenbaum Research Institute, Toronto) for kindly providing the lentiviral vector pLenti-mCherry-NLS. We thank Ralitsa Madsen (MRC PPU Dundee) for advice on validation of genome edited clones.

我们感谢Gillian Smith（Dundee Ninewells医院Jacqui Wood癌症中心）友好地提供Caov-3卵巢癌细胞，以及Daniel Durocher（多伦多Lunenfeld Tannenbaum研究所）友好地提供慢病毒载体pLenti-mCherry NLS。我们感谢Ralitsa Madsen（MRC PPU Dundee）提供有关验证基因组编辑克隆的建议。

We thank all JR lab members especially Ivan Muñoz and Rathan Singh Jadav for useful discussions and Nelma Palminha (currently at Artios Pharma) and Wissem Bououdina for help with blinding of the cell viability assays in Fig. 3. This work was supported by the Medical Research Council (UK; Grant Number MC_UU_00038/5), the European Research Council (ERC Stg No.

。这项工作得到了医学研究理事会（英国；批准号MC\U UU\U 00038/5），欧洲研究理事会（ERC Stg No。

IDRE-715127) and Cancer Research UK (CDF C57404/A21782).Author informationAuthors and AffiliationsMRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, DD1 5EH, UKVikram Tejwani, Thomas Carroll, Thomas Macartney, Giulia Saredi, Rachel Toth & John RouseDivision of Molecular, Cell and Developmental Biology, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, DD1 5EH, UKSusanne Bandau & Constance AlabertAuthorsVikram TejwaniView author publicationsYou can also search for this author in.

IDRE-715127）和英国癌症研究（CDF C57404/A21782）。作者信息作者和附属机构邓迪大学，邓迪，DD1 5EH，UKVikram Tejwani，Thomas Carroll，Thomas Macartney，Giulia Saredi，Rachel Toth＆John RouseDivision of Molecular，Cell and Development Biology，School of Life Sciences，Wellcome Trust Biocentre，邓迪大学，邓迪，DD1 5EH，UKSusanne Bandau＆Constance AlabertAuthorsVikram TejwaniView作者出版物你也可以在中搜索这位作者。

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PubMed Google ScholarContributionsV.T. executed all the experiments and J.R. conceived the study. T.C. helped with analysis of microscopy data. T.M. generated DNA constructs for genome editing, and R.T. generated other DNA constructs. S.B., C.A. and G.S. helped with high content ScanR imaging and data analysis.Corresponding authorCorrespondence to.

PubMed谷歌学术贡献SV。T、进行了所有实验，J.R.构思了这项研究。T、 C.帮助分析显微镜数据。T、 M.生成了用于基因组编辑的DNA构建体，R.T.生成了其他DNA构建体。S、。对应作者对应。

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Reprints and permissionsAbout this articleCite this articleTejwani, V., Carroll, T., Macartney, T. et al. PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability.

转载和许可本文引用本文Tejwani，V.，Carroll，T.，Macartney，T。等人。PROTAC介导的WRN解旋酶的条件性降解是选择性杀死具有微卫星不稳定性的癌细胞的潜在策略。

Sci Rep 14, 20824 (2024). https://doi.org/10.1038/s41598-024-71160-5Download citationReceived: 05 June 2024Accepted: 26 August 2024Published: 06 September 2024DOI: https://doi.org/10.1038/s41598-024-71160-5Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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KeywordsWRNWerner syndromeMSIMicrosatellite instabilityCancerPROTACDegrader

关键词WRNWerner综合征MSI微卫星不稳定性癌症PROTACDegrader

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