PO.ET03.05 · 实验与分子治疗
来源于患者细胞系的TKI诱导性药物耐受持留细胞的特征分析
Characterization of TKI-induced drug-tolerant persister cells from patient-derived cell lines
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：靶向治疗为癌基因驱动的癌症带来了显著的临床获益，但复发仍不可避免。越来越多的证据表明，一小群药物耐受持留（DTP）细胞在初始治疗后存活下来，并最终导致耐药的产生。因此，了解这些DTP细胞的易感性对于开发能够在耐药出现之前加以预防的策略至关重要。
材料与方法：患者来源细胞系（PDC）建立自纳入两项前瞻性临床试验的NSCLC患者：MATCH-R研究（2015至2022年）和正在进行的STING试验。使用的两个主要模型为MR57（携带EML4-ALK融合及ALK C1156Y/G1269A突变，对第3代ALK抑制剂lorlatinib敏感）和ST6566（携带EGFR L858R突变，对osimertinib高度敏感）。
结果与讨论：DTP细胞的特征是显著的表型可塑性。在两个PDC模型中，我们观察到上皮和间充质标志物的共表达，支持一种杂合EMT状态。这种可塑性得到BRD4（调控EMT相关转录程序）以及FGFR信号（通过通路重连）的支撑。Phospho-RTK和磷酸激酶芯片显示IGF1R和STAT3磷酸化水平升高，与DTP细胞中的适应性信号传导一致。同时，相较于初治细胞，DTP细胞表现出升高的gammaH2AX焦点，与TKI诱导的基因组不稳定性一致，后者可能诱导对DNA损伤反应通路的依赖性。体外模型还能够利用长期生存读数功能性地评估这些特征以及其他关键的DTP细胞标志，包括结晶紫克隆形成实验和IncuCyte活细胞成像。这些实验揭示，用BRD4抑制剂JQ1靶向EMT相关转录调控、用erdafitinib抑制FGFR驱动的信号，或通过ATM抑制剂AZD-0156阻断DNA损伤修复，均可显著延迟DTP细胞的再生长。总之，这些发现凸显了DTP状态期间出现的多个可干预的弱点。
结论：总体而言，我们的结果证明DTP细胞的再生长在体外可被延迟，提示存在可利用的易感性。尽管仍需进一步验证，但这些结果为在体内检验这些策略提供了理论依据，最终目标是为患者治疗方案提供指导。
查看英文原文 English abstract
Introduction: Targeted therapies provide substantial clinical benefit in oncogene-driven cancers, yet relapse remains inevitable. Increasing evidence suggests that a small population of drug-tolerant persister (DTP) cells survives initial therapy and ultimately gives rise to resistance. Understanding the vulnerabilities of these DTP cells is therefore essential for developing strategies that could prevent resistance before it emerges.
Materials and Methods: Patients-derived cell lines (PDC) were established from NSCLC patients included in two prospective clinical trials: MATCH-R study (2015 to 2022) and the ongoing STING trial. The two main models used were MR57, harboring EML4-ALK fusion and ALK C1156Y/G1269A mutation and sensitive to the 3rd generation ALK inhibitor lorlatinib, and ST6566, carrying an EGFR L858R mutation and highly sensitive to osimertinib.
Results and Discussion: DTP cells are characterized by marked phenotypic plasticity. In two PDC models, we observed the co-expression of epithelial and mesenchymal markers, supporting a hybrid EMT state. This plasticity is supported by BRD4, which regulates EMT-associated transcriptional programs, and by FGFR signaling, through pathway rewiring. Phospho-RTK and phosphokinase arrays revealed increased IGF1R and STAT3 phosphorylation level, consistent with adaptative signaling in DTP cells. In parallel, DTP cells displayed elevated gammaH2AX foci relative to treatment-naive cells, consistent with TKI-induced genomic instability that may induce a dependency on DNA-damage response pathways. In vitro models also enable to functionally assess these features, as well as other key DTP cells hallmarks, using long-term survival readouts, including crystal-violet clonogenic assays and IncuCyte live-cell imaging. These experiments revealed that targeting EMT-associated transcriptional control with the BRD4 inhibitor JQ1, inhibiting FGFR-driven signaling with erdafitinib, or blocking DNA-damage repair via the ATM inhibitor AZD-0156 each significantly delayed DTP regrowth. Together, these findings underline multiple actionable weaknesses emerging during the DTP state.
Conclusion: Overall, our results demonstrate that DTP cells regrowth can be delayed in vitro , suggesting exploitable vulnerabilities. While further validation is required, they provide a rational for testing these strategies in vivo , with an ultimate goal of informing patient therapeutic approach.
利益披露 Disclosure
F. Braye, None..
I. Alonso Garcia, None..
V. Boursier, None.