PO.CL06.02 · 临床研究
高通量筛选鉴定横纹肌肉瘤中CDK8/19抑制剂与DHFR抑制剂之间的协同药物相互作用
High-throughput screening identifies synergistic drug interactions between CDK8/19 and DHFR inhibitors in rhabdomyosarcoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
横纹肌肉瘤（RMS）是一种侵袭性的儿童软组织肉瘤，具有显著的治疗相关并发症和不良结局，尤其是在转移性或复发性疾病中。尽管诊断和治疗方法已有改善，但对于具有高危分子特征（包括携带PAX3::FOXO1融合的肿瘤）的患者，生存率仍然较低。该融合蛋白驱动一种异常的转录程序，促进肿瘤进展和治疗耐药，凸显了对新治疗策略的需求。在本研究中，我们探究了通过抑制转录调控激酶CDK8/19所产生的治疗脆弱性。利用高通量药物组合筛选，我们针对一个包含2,803种临床前、研究性和已批准小分子的化合物库测试了数种CDK8/19抑制剂。该筛选鉴定出CDK8/19抑制剂与二氢叶酸还原酶（DHFR）抑制剂之间存在强烈的协同相互作用。无监督层次聚类显示，多种DHFR抑制剂形成一个独特的聚类，具有高度相似的协同模式，支持这一相互作用的稳健性。后续的细胞活力实验证实，在PAX3::FOXO1阳性和融合阴性的RMS细胞系中均存在协同作用。为界定这一协同作用的生物学基础，我们检测了其对转录调控、细胞周期动态和凋亡的影响。Annexin-V/DAPI染色表明，联合治疗产生的凋亡和G1期阻滞显著高于任一单药。EdU掺入实验显示DNA复制显著减少，提示核苷酸合成受损。转录组学分析（包括基因集富集分析）揭示，CDK8/19抑制剂上调PAX3::FOXO1融合靶基因、MYC调控基因和氧化磷酸化通路，而DHFR抑制剂主要诱导免疫相关和KRAS信号程序。每类药物的转录反应在很大程度上各不相同，而联合治疗逆转了单药所驱动的表达模式。重要的是，DHFR抑制触发嘧啶生物合成基因的代偿性激活，而这一反应被CDK8/19抑制所抑制，为观察到的协同作用提供了机制基础。ChIP-seq和蛋白质组学数据进一步支持了这些发现。此外，CDK8/19抑制放大了DHFR抑制剂诱导的DNA损伤，表现为gammaH2AX（γH2AX）积累升高。总体而言，这些结果凸显了一种同时破坏PAX3::FOXO1驱动的转录程序和核苷酸依赖性DNA修复的治疗策略。因此，CDK8/19抑制剂与DHFR抑制剂的联合可能克服耐药机制并增强RMS的治疗疗效。
查看英文原文 English abstract
Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue sarcoma marked by substantial treatment-related morbidity and poor outcomes, particularly in metastatic or recurrent disease. Although diagnostic and therapeutic approaches have improved, survival remains low for patients with high-risk molecular features, including tumors harboring the PAX3::FOXO1 fusion. This fusion protein drives an aberrant transcriptional program that promotes tumor progression and resistance to therapy, underscoring the need for new treatment strategies. In this work, we investigated therapeutic vulnerabilities created by inhibiting the transcription-regulatory kinases CDK8/19. Using a high-throughput drug combination screen, we tested several CDK8/19 inhibitors against a library of 2,803 preclinical, investigational, and approved small molecules. The screen identified a strong synergistic interaction between CDK8/19 inhibitors and dihydrofolate reductase (DHFR) inhibitors. Unsupervised hierarchical clustering showed that multiple DHFR inhibitors formed a distinct cluster with highly similar patterns of synergy, supporting the robustness of this interaction. Follow-up viability assays confirmed synergy in both PAX3::FOXO1-positive and fusion-negative RMS cell lines. To define the biological basis of this synergy, we examined effects on transcriptional regulation, cell-cycle dynamics, and apoptosis. Annexin-V/DAPI staining demonstrated that combined treatment produced significantly higher apoptosis and G1 arrest than either drug alone. EdU incorporation assays showed a strong decrease in DNA replication, indicating impaired nucleotide synthesis. Transcriptomic profiling, including gene set enrichment analysis, revealed that CDK8/19 inhibitors upregulated PAX3::FOXO1 fusion targets, MYC-regulated genes, and oxidative phosphorylation pathways, whereas DHFR inhibitors primarily induced immune-related and KRAS signaling programs. The transcriptional responses to each drug class were largely distinct, and combination treatment reversed expression patterns driven by single agents. Importantly, DHFR inhibition triggered compensatory activation of pyrimidine biosynthesis genes, a response that was suppressed by CDK8/19 inhibition, providing a mechanistic basis for the observed synergy. ChIP-seq and proteomic data further supported these findings. Additionally, CDK8/19 inhibition amplified DHFR inhibitor-induced DNA damage, reflected by elevated gammaH2AX accumulation. Overall, these results highlight a therapeutic strategy that concurrently disrupts PAX3::FOXO1-driven transcriptional programs and nucleotide-dependent DNA repair. The combination of CDK8/19 and DHFR inhibitors may therefore overcome resistance mechanisms and enhance treatment efficacy in RMS.
利益披露 Disclosure
U. Jo, None.