Unraveling Resistance Mechanisms to the Novel Nucleoside Analog RX-3117 in lung cancer: Insights into DNA Repair, Cell Cycle Dysregulation and Targeting PKMYT1 for Improved Therapy

Background. Nucleoside analogues are crucial in treating non-small cell lung cancer (NSCLC), but resistance hampers patient outcomes. RX-3117—a cytidine analogue— shows promise in gemcitabine-resistant cancers, yet mechanisms underlying acquired resistance to this drug remain unexplored. This study includes a comprehensive investigation into RX-3117 resistance mechanisms by leveraging new preclinical models and cutting-edge genomic tools, including a genome-wide CRISPR-Cas9 knockout screen and transcriptomics. Methods. NSCLC cell lines A549 and SW1573 were exposed to stepwise increasing concentrations of RX-3117 to establish stable resistant subclones, confirmed by SRB and clonogenic assays. Intracellular RX-3117 nucleotide levels were measured via LC/MS-MS, prompting the evaluation and modulation of the expression of key metabolic enzymes by Western blot and siRNA. A CRISPR-Cas9 screen identified genes whose loss increased RX- 3117 sensitivity, while RNA-sequencing with differential expression analyses revealed resistance-related pathways, further investigated through cell cycle distribution, knock-out, and ELISA assays. Results. Resistant clones exhibited decreased accumulation of RX-3117 nucleotides, which however, was not associated to reduced expression of activation enzymes (UCK2, UMPK, CMPK, NME1/NDPK, RR1 and RR2). Instead, increased expression was observed in certain DNA repair and deactivation enzymes (NT5C3) but pharmacological inhibition and silencing of the latter did not circumvent resistance. Remarkably, a comprehensive approach with CRISPR-Cas9 screen highlighted DNA-repair and cell cycle determinants as key sensitizing genes. XL-PCR and RNA-sequencing confirmed aberrations in DNA-repair and pathways involved in cell cycle regulation. Knock-out and pharmacological inhibition validated the role of PKMYT1, a protein kinase involved in G2/M transition and genomic stability. RX-3117- resistant A549 cells showed enhanced sensitivity to the PKMYT1 inhibitor lunresertib and its synergism with RX-3117, suggesting further studies, especially in patients with high PKMYT1 expression who have significantly shorter survival rates, as observed in public databases and validated in an internal cohort of NSCLC patients. Conclusion. By integrating CRISPR-Cas9 technology with functional assays and transcriptomics, our study established a framework for decoding resistance mechanisms and highlights potential therapeutic strategies to enhance RX-3117 efficacy in NSCLC. We demonstrated for the first time that aberrant DNA repair and cell cycle dysregulation led resistance, identifying PKMYT1 as a promising target. Overall design: RNA-seq profiling of A549/H460/SW1573 cells and their RX-3117 resistance derivatives.

研究背景
核苷类似物（nucleoside analogues）是非小细胞肺癌（non-small cell lung cancer, NSCLC）治疗的关键用药，但耐药性会严重影响患者预后。RX-3117作为一种胞苷类似物（cytidine analogue），在吉西他滨（gemcitabine）耐药性癌症中展现出应用潜力，但目前针对该药物的获得性耐药机制仍未被阐明。本研究借助全新临床前模型与前沿基因组学工具——包括全基因组CRISPR-Cas9敲除筛选与转录组学技术——对RX-3117的耐药机制开展了全面探究。

研究方法
我们将非小细胞肺癌细胞系A549与SW1573置于逐步升高浓度的RX-3117环境中培养，以构建稳定的耐药亚克隆，并通过磺酰罗丹明B（SRB）实验与克隆形成实验验证耐药模型的成功建立。采用液相色谱-串联质谱（LC/MS-MS）检测细胞内RX-3117核苷酸的水平，以此为依据通过蛋白质印迹（Western blot）与小干扰RNA（siRNA）技术，对关键代谢酶的表达进行评估与调控。通过CRISPR-Cas9筛选，我们鉴定出了敲除后可增强RX-3117敏感性的基因；同时通过RNA测序与差异表达分析，揭示了与耐药相关的信号通路，并进一步通过细胞周期分布检测、基因敲除实验与酶联免疫吸附测定（ELISA）对相关通路展开验证。

研究结果
耐药克隆细胞内RX-3117核苷酸的蓄积量出现下降，但该现象与药物活化酶（UCK2、UMPK、CMPK、NME1/NDPK、RR1及RR2）的表达下调并无关联。与之相反，部分DNA修复酶与药物失活酶（如NT5C3）的表达水平出现上调，但针对该类酶的药理学抑制与基因沉默均未能逆转耐药性。值得注意的是，CRISPR-Cas9筛选的综合分析结果显示，DNA修复与细胞周期调控相关基因是关键的增敏靶点。交联聚合酶链反应（XL-PCR）与RNA测序结果证实，DNA修复通路与细胞周期调控通路存在异常。基因敲除与药理学抑制实验验证了PKMYT1的功能：该蛋白激酶参与G2/M期转换与基因组稳定性维持。对RX-3117耐药的A549细胞，对PKMYT1抑制剂lunresertib表现出更强的敏感性，且该抑制剂与RX-3117具有协同作用，这提示后续可开展相关研究——尤其是针对PKMYT1高表达的非小细胞肺癌患者：这类患者的生存率显著更低，该结论已在公共数据库中得到验证，并在本研究内部的非小细胞肺癌患者队列中得到确认。

研究结论
本研究将CRISPR-Cas9技术与功能实验、转录组学技术相结合，构建了一套解析耐药机制的研究框架，并提出了可用于提升RX-3117在非小细胞肺癌治疗中疗效的潜在治疗策略。我们首次证实，异常DNA修复与细胞周期失调是导致耐药的原因，并鉴定出PKMYT1作为极具潜力的治疗靶点。

整体实验设计
对A549、H460、SW1573细胞及其RX-3117耐药衍生株进行RNA测序分析。