PO.ET02.07 · 实验与分子治疗
追踪 APOBEC3A 以克服肺癌耐药
Tracking APOBEC3A to overcome drug resistance in lung cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
靶向治疗已彻底改变了晚期非小细胞肺癌（NSCLC）的治疗格局，但获得性耐药仍是一大挑战。我们近期证明，胞苷脱氨酶 APOBEC3A（A3A）可被肺癌靶向治疗诱导，并通过促进耐药持留（DTP）细胞的进化，在推动获得性耐药中发挥关键作用。虽然 A3A 突变特征在 TKI 治疗后的肿瘤中占 70-80%，而在未治疗样本中仅占 20-25%，但一些耐药肿瘤缺乏这些突变。同样，虽然许多肺癌细胞系在 TKI 治疗后上调 A3A 表达，但其他细胞系则不会。我们对治疗诱导的 A3A 表达和活性的肿瘤内和肿瘤间异质性的认识仍然有限。大多数用于研究 A3A 表达和活性的现有方法都是针对整体群体进行的终点检测。由于 A3A 激活是间歇性的，且发生在可检测和量化的基因组变化之前，因此长期以来无法在单细胞水平上随时间解析 A3A 的活性。为克服这些障碍，我们改造了 HiBiT 纳米荧光素酶报告系统，开发了一种在药物处理的肿瘤细胞中实时单细胞监测 A3A 活性的方法。利用 A3A 对 mRNA 发夹底物的偏好，我们将优化的 A3A 底物序列整合到 HiBiT 标签中。在未编辑的构型下，HiBiT 无法与 LgBiT 互补。然而，当 A3A 在 mRNA 发夹环内产生 C>U 编辑时，就会发生互补，由此产生的发光信号即报告 A3A 正在活跃地进行编辑。我们将 A3A-HiBiT 报告系统导入源自患者的 EGFR、ALK 和 KRAS 突变型 NSCLC 细胞系。TKI 治疗诱导了报告基因编辑，这可通过 HiBiT 发光检测到，并经等位基因特异性微滴数字 PCR 验证。在催化失活的 A3A E72A 存在下或在 A3A 缺失的细胞系中未观察到报告基因活性，证实了对 A3A 介导编辑的特异性。为研究靶向治疗处理的肺癌细胞中治疗诱导 A3A 活性的肿瘤内异质性，我们构建了稳定表达 LgBiT 的 A3A-HiBiT 细胞。目前正在开展实验，以优化超灵敏生物发光显微成像，从而在活单细胞中表征和监测 A3A 编辑的间歇性特征，并利用单细胞 RNA-seq 将 A3A-HiBiT 编辑与单细胞转录异质性相关联。总之，我们的 A3A-HiBiT 报告系统提供了一个灵敏、可扩展且可量化的系统，用于实时监测 A3A 活性，为研究 A3A 在肿瘤演化和耐药中的作用提供了新的机会。
查看英文原文 English abstract
Targeted therapies have transformed treatment for advanced non-small cell lung cancer (NSCLC), but acquired resistance remains a challenge. We recently showed that the cytidine deaminase APOBEC3A (A3A) is induced by lung cancer targeted therapies and plays a crucial role in promoting acquired drug resistance by facilitating the evolution of drug-tolerant persister (DTP) cells. While A3A mutational signatures are found in 70-80% of tumors post-TKI treatment versus 20-25% in untreated samples, some resistant tumors lack these mutations. Similarly, while many lung cancer cell lines upregulate A3A expression upon TKI treatment, others do not. Our understanding of intra- and inter-tumoral heterogeneity of therapy-induced A3A expression and activity is limited. Most available methods for studying A3A expression and activity are endpoint assays performed on bulk populations. Because A3A activation is episodic and occurs before detectable genomic changes can be quantified, resolving the activity of A3A in single cells over time has not been possible. To overcome these barriers we adapted the HiBiT nano-luciferase reporter system to develop a method for real-time single-cell monitoring of A3A activity in drug-treated tumor cells.Leveraging the preference of A3A for mRNA hairpin substrates, we incorporated optimized A3A-substrate sequences into the HiBiT tag. In the unedited configuration, HiBiT is unable to complement with LgBiT. However when A3A creates a C>U edit within the mRNA hairpin loop, complementation occurs and the resulting luminescent signal reports that A3A is actively editing. We introduced the A3A-HiBiT reporter into patient-derived EGFR, ALK, and KRAS-mutant NSCLC cell lines. TKI treatment induced reporter editing, as detected by HiBiT luminescence and validated by allele-specific droplet digital PCR. No reporter activity was observed in the presence of catalytically inactive A3A E72A or in cell lines with deletion of A3A, confirming specificity for A3A-mediated editing. To investigate intra-tumoral heterogeneity of therapy-induced A3A activity in lung cancer cells treated with targeted therapies, we generated A3A-HiBiT cells stably expressing LgBiT. Experiments are underway to optimize ultra-sensitive bioluminescent microscopy to characterize and monitor the episodic nature of A3A editing in live single cells, as well as to correlate A3A-HiBiT editing with single cell transcriptional heterogeneity using single cell RNA-seq. In summary, our A3A-HiBiT reporter provides a sensitive, scalable, and quantifiable system for real-time monitoring of A3A activity, enabling new opportunities to study the role of A3A in tumor evolution and drug resistance.
利益披露 Disclosure
M. Di Marco, None.