Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India.
Homi Bhabha National Institute, Anushaktinagar, Mumbai, India.
Aging Cell. 2022 Jun;21(6):e13625. doi: 10.1111/acel.13625. Epub 2022 May 18.
Mutation in Werner (WRN) RECQL helicase is associated with premature aging syndrome (Werner syndrome, WS) and predisposition to multiple cancers. In patients with solid cancers, deficiency of the WRN RECQL helicase is paradoxically associated with enhanced overall survival in response to treatment with TOP1 inhibitors, which stabilize pathological TOP1-DNA-covalent-complexes (TOP1cc) on the genome. However, the underlying mechanism of WRN in development of chemoresistance to TOP1 inhibitors is not yet explored. Our whole-genome transcriptomic analysis for ~25,000 genes showed robust activation of NF-κB-dependent prosurvival genes in response to TOP1cc. CRISPR-Cas9 knockout, shRNA silencing, and under-expression of WRN confer high-sensitivity of multiple cancers to TOP1 inhibitor. We demonstrated that WRN orchestrates TOP1cc repair through proteasome-dependent and proteasome-independent process, unleashing robust ssDNA generation. This in turn ensues signal transduction for CHK1 mediated NF-κB-activation through IκBα-degradation and nuclear localization of p65 protein. Intriguingly, our site-directed mutagenesis and rescue experiments revealed that neither RECQL-helicase nor DNA-exonuclease enzyme activity of WRN (WRN , WRN , and WRN ) were required for TOP1cc removal, ssDNA generation and signaling for NF-κB activation. In correlation with patient data and above results, the TOP1 inhibitor-based targeted therapy showed that WRN-deficient melanoma tumors were highly sensitive to TOP1 inhibition in preclinical in vivo mouse model. Collectively, our findings identify hitherto unknown non-enzymatic role of WRN RECQL helicase in pathological mechanisms underlying TOP1cc processing and subsequent NF-κB-activation, offering a potential targeted therapy for WRN-deficient cancer patients.
Werner（WRN）RECQL 解旋酶的突变与早老综合征（Werner 综合征，WS）和多种癌症易感性相关。在实体瘤患者中，WRN RECQL 解旋酶的缺失与对 TOP1 抑制剂治疗的总体生存改善呈悖论关系，TOP1 抑制剂可稳定基因组上的病理性 TOP1-DNA 共价复合物（TOP1cc）。然而，WRN 在发展对 TOP1 抑制剂的化学耐药性中的作用机制尚未得到探索。我们对约 25000 个基因的全基因组转录组分析显示，TOP1cc 反应强烈激活 NF-κB 依赖性生存相关基因。CRISPR-Cas9 敲除、shRNA 沉默和 WRN 低表达使多种癌症对 TOP1 抑制剂高度敏感。我们证明 WRN 通过蛋白酶体依赖和非依赖的过程来协调 TOP1cc 的修复，释放出大量的单链 DNA。这反过来又通过 IκBα 降解和 p65 蛋白的核定位引发 CHK1 介导的 NF-κB 激活的信号转导。有趣的是，我们的定点突变和挽救实验表明，WRN 的 RECQL 解旋酶或 DNA 外切酶活性（WRN 、WRN 和 WRN ）均不是 TOP1cc 去除、ssDNA 生成和 NF-κB 激活信号所必需的。与患者数据和上述结果相关，基于 TOP1 抑制剂的靶向治疗表明，WRN 缺陷型黑色素瘤肿瘤在临床前体内小鼠模型中对 TOP1 抑制高度敏感。总之，我们的研究结果确定了 WRN RECQL 解旋酶在 TOP1cc 加工和随后的 NF-κB 激活的病理机制中以前未知的非酶活性，为 WRN 缺陷型癌症患者提供了一种潜在的靶向治疗方法。
