PO.ET03.02 · 实验与分子治疗
手术刀下的WRN：HCT116和RKO中解旋酶结构域热点及剪接位点敲入
WRN under the scalpel: Helicase-domain hotspot & splice-site knock-ins in HCT116 and RKO
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
WRN解旋酶是微卫星高度不稳定（MSI-H）肿瘤中的一个合成致死脆弱靶点，然而随着小分子WRN抑制剂的推进，等位基因特异性耐药是一个可预见的风险。在天然调控背景下构建的内源性点突变模型，比过表达系统更能忠实地反映药物-靶点结合和通路补偿。此类模型能够进行严格的作用机制确认、耐药隐患图谱绘制、化学骨架备选选择、患者分层/生物标志物假设，以及更快的构效关系（SAR）和联合方案设计。在同源的HCT116和RKO模型中，两种WRN靶向化学骨架在特定的隐患节点上出现分歧。WT HCT116高度敏感（VVD-214为48.47 nM；HRO761为86.89 nM），基线RKO仍有反应（366/720 nM）。C727决定VVD-214耐药：C727A使VVD-214升至>10 µM，同时保持接近WT的HRO761（153-179 nM）；C727S使HRO761升至2.1-3.5 µM，VVD-214升至>10 µM；在RKO-C727S中，两者均超过10 µM。I852F表现出相反的模式——VVD-214仍保持强效（62-77 nM），而HRO761失活（>10 µM）。G729D和F730L导致双侧右移，对HRO761的右移更大（VVD-214为0.71-1.34 µM；HRO761为2.9-8.0 µM）；联合G729D+I852F使HRO761失效（>10 µM），而VVD-214仍保持亚µM水平（0.78-0.82 µM）。剪接变体c.1577-1G>C对VVD-214高度敏感（36-37 nM），但对HRO761右移（约1.11 µM）。总体而言，C727→VVD-214耐药，I852→HRO761耐药，而G729/F730/剪接则赋予更广泛的HRO761偏向性——为化学骨架切换和备选选择提供指导。等位基因通过CRISPR-Cas9 HDR使用约200-nt的ssODN供体导入；单细胞克隆经序列验证（Sanger/NGS）、STR鉴定认证并经支原体阴性冻存。剂量-反应活力实验（72-96小时，10点曲线）用四参数逻辑斯蒂模型拟合以推导IC₅₀；每个克隆包括n≥2-3个生物学重复。该WRN等位基因组合可立即部署用于：（1）构建等位基因分辨率的耐药图谱，（2）基于C727/I852的互补性优先选择化学骨架备选，（3）为MSI-H情境生成患者选择/生物标志物假设，以及（4）加速SAR和联合策略——从而为VVD-214类和HRO761类WRN抑制剂前置化学和临床降风险工作。
查看英文原文 English abstract
The WRN helicase is a synthetic-lethal vulnerability in microsatellite-instability-high (MSI-H) tumors, yet allele-specific resistance is a foreseeable risk as small-molecule WRN inhibitors advance. Endogenous point-mutation models, built in the native regulatory context, more faithfully capture drug-target engagement and pathway compensation than overexpression systems. Such models enable rigorous mechanism-of-action confirmation, resistance-liability mapping, chemotype backup selection, patient-stratification/biomarker hypotheses, and faster SAR and combination design.Across isogenic HCT116 and RKO models, two WRN-targeting chemotypes diverged at specific liability nodes. WT HCT116 was highly sensitive (VVD-214 48.47 nM; HRO761 86.89 nM), and baseline RKO remained responsive (366/720 nM). C727 dictated VVD-214 resistance: C727A drove VVD-214 to >10 µM while preserving near-WT HRO761 (153-179 nM); C727S pushed HRO761 to 2.1-3.5 µM and VVD-214 to >10 µM; in RKO-C727S, both exceeded 10 µM. I852F showed the reciprocal pattern-VVD-214 stayed potent (62-77 nM) but HRO761 was inactive (>10 µM). G729D and F730L caused bilateral right-shifts, larger for HRO761 (VVD-214 0.71-1.34 µM; HRO761 2.9-8.0 µM); combining G729D+I852F abolished HRO761 (>10 µM) while VVD-214 remained sub-µM (0.78-0.82 µM). The splice variant c.1577-1G>C was hypersensitive to VVD-214 (36-37 nM) yet right-shifted for HRO761 (~1.11 µM). Collectively, C727 → VVD-214 resistance and I852 →HRO761 resistance, while G729/F730/splice confer a broader HRO761 bias-guiding chemotype switching and backup selection.Alleles were introduced by CRISPR-Cas9 HDR using ~200-nt ssODN donors; single-cell clones were sequence-verified (Sanger/NGS), STR-authenticated, and mycoplasma-free banked. Dose-response viability assays (72-96 h, 10-point curves) were fit with four-parameter logistic models to derive IC₅₀; each clone included n≥2-3 biological replicates. This WRN allele panel is immediately deployable to (1) build allele-resolved resistance maps, (2) prioritize chemotype backups based on the C727/I852 complementarity, (3) generate patient-selection/biomarker hypotheses for MSI-H settings, and (4) accelerate SAR and combination strategies-thereby front-loading chemical and clinical derisking for VVD-214- and HRO761-class WRN inhibitors.
利益披露 Disclosure
Y. Huang,
Kyinno Biotechnology Co., LTD Employment.
X. Gou,
Kyinno Biotechnology Co., LTD Employment.
J. Ning,
Kyinno Biotechnology Co., LTD Employment.
F. Hao,
Kyinno Biotechnology Co., LTD Employment.