PO.ET04.01 · 实验与分子治疗
体内腺嘌呤碱基编辑STK11 Q37*重编程肿瘤以产生放射增敏效应
In vivo adenine base editing of STK11 Q37* reprograms tumor for radiosensitizing effect
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
本研究旨在探讨使用AAV介导的腺嘌呤碱基编辑器（ABE）递送进行体内基因治疗，对携带STK11无义突变STK11 Q37*（c.109C>T）的非小细胞肺癌（NSCLC）的治疗潜力，并进一步探索其影响放射敏感性的分子机制。放疗是局部晚期或不可手术NSCLC患者的标准治疗，由促肿瘤体细胞突变驱动的放射抗性严重限制临床疗效，凸显了对诸如精确基因校正等靶向策略以克服这一障碍的迫切需求。通过基于CRISPR的体内突变文库筛选结合全外显子组测序，我们鉴定STK11 Q37*为放射抗性的关键驱动因素。为恢复STK11功能，我们工程改造了一组具有不同PAM/TAM兼容性的ABE，并进一步开发了一种高保真变体A8E-N108Q-R26G，能够精确校正致病腺嘌呤而无旁观者编辑。使用稳定携带STK11 Q37*位点的HEK293T报告细胞，我们发现spCas9-A8EQR（N108Q-R26G）实现了最高的总体校正效率，以卓越保真度修复多达60%的突变等位基因。随后将优化的编辑器包装进双AAV系统，体内递送至荷皮下患者来源类器官异种移植的人源化小鼠。AAV介导的碱基编辑与放疗联合产生协同抗肿瘤效应，并显著抑制肿瘤进展。机制研究揭示，STK11 Q37*的精确校正恢复了内源性LKB1蛋白表达和激酶活性，重新激活维持氧化还原稳态所需的下游信号。恢复的LKB1通过限制转录调节因子BACH1的泛素化和蛋白酶体降解，直接稳定BACH1，从而保持BACH1介导的对抗氧化基因程序的抑制。同时，LKB1再表达抑制NRF2核内积累和转录活性，导致NRF2驱动的解毒和抗氧化通路表达降低。这种协调调控显著提升了照射后的胞内ROS水平，并重建放射诱导的细胞毒性应激，共同增强肿瘤放射敏感性。总之，我们的研究将STK11 Q37*鉴定为放射抗性的可治疗靶向驱动因素，并证明精确的ABE介导校正提供了一种有前景的基于基因编辑的策略，以改善NSCLC的治疗结局。
查看英文原文 English abstract
In this study, we aim to investigate the therapeutic potential of in vivo genetic therapy using AAV-mediated adenine base editor (ABE) delivery for non-small cell lung cancer (NSCLC) carrying the STK11 nonsense mutation STK11 Q37* (c.109C>T), and further explore the molecular mechanisms underlying its impact on radiosensitivity. Radiotherapy is a standard treatment for locally advanced or inoperable NSCLC patients, radiation resistance driven by tumor-promoting somatic mutations severely limits clinical efficacy, underscoring the urgent need for targeted strategies such as precise gene correction to overcome this barrier. Through CRISPR-based in vivo mutation library screening combined with whole-exome sequencing, we identified STK11 Q37* as a critical driver of radiation resistance. To restore STK11 function, we engineered a panel of ABEs with distinct PAM/TAM compatibilities and further developed a high-fidelity variant, A8E-N108Q-R26G, which enables precise correction of the pathogenic adenine without bystander editing. Using HEK293T reporter cells stably harboring the STK11 Q37* locus, we found that spCas9-A8EQR (N108Q-R26G) achieved the highest overall correction efficiency, repairing up to 60% of mutant alleles with superior fidelity. The optimized editor was subsequently packaged into a dual-AAV system and delivered in vivo to humanized mice bearing subcutaneous patient-derived organoid xenografts. AAV-mediated base editing combined with radiotherapy produced synergistic antitumor effects and significantly suppressed tumor progression. Mechanistic studies revealed that precise correction of STK11 Q37* restores endogenous LKB1 protein expression and kinase activity, reactivating downstream signaling required for maintaining redox homeostasis. Restored LKB1 directly stabilizes the transcriptional regulator BACH1 by limiting its ubiquitination and proteasomal degradation, thereby preserving BACH1-mediated repression of antioxidant gene programs. Concurrently, LKB1 re-expression dampens NRF2 nuclear accumulation and transcriptional activity, leading to reduced expression of NRF2 -driven detoxification and antioxidant pathways. This coordinated regulation markedly elevates intracellular ROS levels following irradiation and reinstates radiation-induced cytotoxic stress, collectively enhancing tumor radiosensitivity. In summary, our study identifies STK11 Q37* as a therapeutically actionable driver of radiation resistance and demonstrates that precise ABE-mediated correction provides a promising gene-editing-based strategy to improve treatment outcomes in NSCLC.
利益披露 Disclosure
J. Yan, None..
Y. Xu, None..
Q. Fang, None..
J. Yu, None..
Z. Yuan, None.