PO.ET03.05 · 实验与分子治疗
ecDNA介导的癌基因扩增是NSCLC对EGFR TKI耐药的基础
EcDNA-mediated oncogene amplification underlies EGFR TKI resistance in NSCLC
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
染色体外DNA（ecDNA）驱动多种癌症中的癌基因扩增和治疗耐药，但其对EGFR突变型非小细胞肺癌（NSCLC）中EGFR抑制剂（EGFRi）获得性耐药的作用尚未明确界定。尽管EGFR TKI耐药常通过获得EGFR T790M突变而产生，但许多病例仍无法解释。因此，我们旨在确定ecDNA是否代表一种与EGFR TKI耐药相关的额外机制。我们分析了来自TCGA、PCAWG和Hartwig医学基金会的全基因组测序数据集，涵盖536例NSCLC肿瘤，并使用AmpliconArchitect检测ecDNA结构。在这些公共队列中，与原发肿瘤（14%，20/147；OR≈6.35，p<0.001）和晚期未治疗肿瘤（25%，92/369；OR≈3.01，p=0.02）相比，EGFR TKI治疗肿瘤中ecDNA的患病率显著升高（50%，10/20），提示EGFR TKI暴露与ecDNA患病率升高之间存在显著关联。值得注意的是，在EGFR TKI治疗组中检出的所有ecDNA均携带致癌驱动基因，凸显其潜在的功能相关性。为研究这种富集是否反映耐药相关事件，我们通过从单个EGFR TKI敏感克隆经长期厄洛替尼暴露后衍生的耐药亚克隆生成WGS和RNA-seq数据，建立了一个同基因PC9耐药模型。对25个耐药PC9样本的分析显示约20%的克隆（5/25）获得了ecDNA，且全部含有癌基因。这些ecDNA阳性克隆与EGFR T790M阳性样本（同样为20%，5/25）相互排斥，提示ecDNA的出现代表一条替代性耐药途径，而非T790M下游的继发事件。功能分析证明ecDNA阳性耐药细胞中EGFR下游信号通路被激活。例如，在一个携带新形成的RAF1 ecDNA（拷贝数约24）的耐药亚克隆中，我们观察到RAF1过表达超过100倍以及显著的MAPK/ERK通路激活，GSEA亦支持这一点（NES=1.39，p<0.01）。RAF1抑制的功能实验进一步证实，抑制RAF1驱动的信号可恢复厄洛替尼敏感性，为ecDNA介导的RAF1扩增导致EGFR非依赖性MAPK通路激活提供了有力证据。ecDNA可能通过启用绕过EGFR活性的替代信号通路来促进EGFR TKI耐药的发展。ecDNA介导的癌基因扩增是EGFR TKI耐药NSCLC的一个重要特征，也是潜在的治疗易感性靶点，为旨在克服ecDNA驱动耐药的策略奠定了基础。
查看英文原文 English abstract
Extrachromosomal DNA (ecDNA) drives oncogene amplification and therapeutic resistance in multiple cancers, but its contribution to acquired resistance to EGFR inhibitors (EGFRi) in EGFR-mutant non-small cell lung cancer (NSCLC) is not well defined. While EGFR TKI resistance frequently arises through acquisition of the EGFR T790M mutation, many cases remain unexplained. We therefore aimed to determine whether ecDNA represents an additional mechanism associated with EGFR TKI resistance. We analyzed whole-genome sequencing datasets from TCGA, PCAWG, and the Hartwig Medical Foundation, encompassing 536 NSCLC tumors, and detected ecDNA structures using AmpliconArchitect. Across these public cohorts, ecDNA prevalence was markedly elevated in EGFR TKI-treated tumors (50%, 10/20) compared with primary tumors (14%, 20/147; OR≈6.35, p<0.001) and advanced untreated tumors (25%, 92/369; OR≈3.01, p=0.02), indicating a significant association between EGFR TKI exposure and increased ecDNA prevalence. Notably, all ecDNAs detected in the EGFR TKI-treated group harbored oncogenic drivers, underscoring their potential functional relevance. To investigate whether this enrichment reflects resistance-associated events, we established an isogenic PC9 resistance model by generating WGS and RNA-seq data from resistant subclones derived from a single EGFR TKI-sensitive clone following long-term erlotinib exposure. Analysis of 25 resistant PC9 samples revealed ecDNA acquisition in approximately 20% of clones (5/25), all of which contained oncogenes. These ecDNA-positive clones were mutually exclusive with EGFR T790M-positive samples (also 20%, 5/25), suggesting that ecDNA emergence represents an alternative resistance route rather than a secondary event downstream of T790M. Functional analyses demonstrated activation of EGFR downstream signaling pathways in ecDNA-positive resistant cells. For example, in one resistant subclone harboring a newly formed RAF1 ecDNA (copy number ~24), we observed over 100-fold RAF1 overexpression and marked MAPK/ERK pathway activation, as supported by GSEA (NES = 1.39, p < 0.01). Functional RAF1-inhibition experiments further confirmed that suppressing RAF1-driven signaling restored erlotinib sensitivity, providing strong evidence that ecDNA-mediated RAF1 amplification leads to EGFR-independent MAPK pathway activation. EcDNA may promote the development of EGFR TKI resistance by enabling alternative signaling pathways that bypass EGFR activity. EcDNA-mediated oncogene amplification represents an important feature of EGFR TKI-resistant NSCLC and a potential therapeutic vulnerability, providing a foundation for strategies aimed at overcoming ecDNA-driven resistance.
利益披露 Disclosure
B. Kim, None..
S. Kim, None..
J. Cho, None..
H. Kim, None.