PO.TB09.02 · 肿瘤生物学
KRAS等位基因失衡通过选择性克隆增生和染色体不稳定性重塑NSCLC的肿瘤演进
KRAS allelic imbalance reshapes tumor evolution through selective clonal outgrowth and chromosomal instability in NSCLC
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
KRAS突变型非小细胞肺癌(NSCLC)约占所有NSCLC病例的25%，由于其显著的演化适应性和易于产生耐药的倾向，仍是最难治的实体瘤之一。KRAS等位基因失衡，包括杂合性缺失(LOH)，在患者肿瘤中反复出现，但其在塑造克隆选择和肿瘤演进中的功能作用仍不明确。我们假设，不同亚群携带由竞争适应度驱动的KRAS LOH，并获得独特的基因组事件。为研究KRAS等位基因失衡如何影响肿瘤演化轨迹，我们开发了一种体细胞嵌合基因工程小鼠模型(smGEMM)，该模型整合了一个200基因的CRISPR-Cas9-GFP扰动文库与一个嵌入小鼠6号染色体的等位基因解析荧光报告基因(Tdtomato)，其距Kras^WT约30 Mb，并与Kras^LSL-G12D呈反式排列。每个扰动由与独特捕获序列偶联的sgRNA编码，使得组合的sgRNA-捕获元件可作为可回收的分子条形码用于克隆追踪。该平台允许同时读出等位基因构型和竞争适应度，便于在体内追踪Kras杂合型(TdT⁺/GFP⁺)和Kras-LOH(TdT⁻/GFP⁺)亚克隆。初步的谱系解析分析揭示，在各生物学重复中Kras-LOH亚克隆呈现一致且可重现的选择性扩增。LOH亚克隆持续形成更大的肿瘤并超越杂合群体，表明等位基因失衡赋予了潜在的竞争优势，而非代表中性漂变。我们对报告基因定位的亚克隆进行的全基因组分析显示，Kras-LOH肿瘤经历广泛的基因组重排，包括大范围的拷贝数改变、结构变异和染色体碎裂事件。我们的研究结果表明，基因组规模的不稳定性作为一种被正向选择的性状，在LOH亚克隆的竞争性扩增过程中出现。我们的研究将Kras等位基因失衡确定为克隆演进的活跃驱动因素，将选择优势、克隆扩增和日益增加的基因组复杂性联系起来。通过将等位基因特异性荧光追踪与聚焦的CRISPR扰动文库相结合，我们揭示了Kras突变型NSCLC演化的机制性见解，并揭示了可用于预防或克服KRAS驱动的肺癌耐药的演化脆弱性。
查看英文原文 English abstract
KRAS -mutant non-small cell lung cancer (NSCLC), which accounts ~25% of all NSCLC cases, remains one of the most refractory solid tumors due to its pronounced evolutionary adaptability and propensity to develop resistance. KRAS allelic imbalance, including loss of heterozygosity (LOH), is recurrently observed in patient tumors, its functional role in shaping clonal selection and tumor evolution remains unclear.We hypothesize that distinct subpopulations harbors LOH of KRAS driven by competitive fitness and acquire unique genomic events. To investigate how KRAS allelic imbalance influences tumor evolutionary trajectories, we developed a somatic mosaic genetically engineered mouse model (smGEMM) that integrates a 200-gene CRISPR-Cas9-GFP perturbation library with an allele-resolving fluorescent reporter (Tdtomato) embedded on murine chromosome 6, ~30 Mb from Kras ^WT and in trans with Kras ^LSL-G12D. Each perturbation is encoded by an sgRNA coupled to a unique capture sequence, allowing the combined sgRNA-capture element to serve as a recoverable molecular barcode for clonal tracing. This platform allows simultaneous readout of allelic configuration and competitive fitness, facilitating in vivo tracking of both Kras -heterozygous (TdT⁺/GFP⁺) and Kras -LOH (TdT⁻/GFP⁺) subclones.Preliminary lineage-resolved analyses revealed a consistent and reproducible selective expansion of Kras -LOH subclones across biological replicates. LOH subclones consistently formed larger tumors and overtook heterozygous populations, demonstrating that allelic imbalance confers a potential competitive advantage rather than representing neutral drift. We performed whole-genome profiling of reporter-mapped subclones showed that Kras -LOH tumors undergo extensive genomic rearrangements, including broad copy-number alterations, structural variations, and chromothripsis events. Our findings indicate that genome-scale instability emerges as a positively selected trait during the competitive expansion of LOH subclones.Our study identifies Kras allelic imbalance as an active driver of clonal evolution, linking selective advantage, clonal expansion, and increasing genomic complexity. By coupling allele-specific fluorescent tracing with a focused, CRISPR perturbation library, we uncover mechanistic insights into Kras -mutant NSCLC evolution and reveal evolutionary vulnerabilities that may be leveraged to prevent or overcome resistance in KRAS -driven lung cancer.
利益披露 Disclosure
L. Zhang, None..
A. Bania, None..
A. Savarese, None..
R. Rinaldi, None..
C. Carlino, None..
L. Perelli, None..
G. Genovese, None.