目标:骨肉瘤,主要影响青少年的高度恶性原发性骨肿瘤,经常对初始化疗产生耐药性,导致转移和有限的治疗选择。我们的研究旨在发现转移性和复发性骨肉瘤的新治疗靶点。
方法:在本研究中,我们证明了调节YAP1调节的谷氨酰胺代谢途径以增强OS对DFMO的反应的潜力。我们最初使用单细胞转录组数据来测量MTAP缺失OS患者中多胺代谢的激活水平。来自复发和非复发患者组织的转录组测序数据进一步证实了这一点。确认进行性OS中多胺代谢的激活。通过高通量药物筛选,我们指出了一种YAP1抑制剂CIL56,作为与DFMO联合治疗策略的有希望的候选人。在体内,我们利用PDX和CDX模型验证了该药物组合的疗效.体外,我们进行了蛋白质印迹分析,qPCR分析,免疫荧光染色,和PuMA实验来监测分子表达的变化,分布,和肿瘤转移能力。我们使用CCK-8和集落形成测定法来评估实验组中细胞的增殖能力。我们使用流式细胞术和活性氧探针观察细胞内ROS和谷氨酰胺代谢的变化。最后,我们将RNA-seq与代谢组学联合应用于鉴定用DFMO和CIL56组合处理的OS细胞中的代谢改变.这使我们能够干预并验证YAP1介导的谷氨酰胺代谢途径在DFMO抗性中的作用。
结果:通过单细胞RNA-seq数据分析,我们确定了聚胺代谢显著上调的晚期OS细胞亚群.通过复发性和非复发性OS组织的转录组学分析进一步证实了这种上调。高通量药物筛选揭示了涉及DFMO和CIL56的有希望的组合策略。DFMO处理抑制了OS细胞中YAP1蛋白的磷酸化,促进核进入并启动YAP1介导的谷氨酰胺代谢途径。这降低了细胞内ROS水平,对抗DFMO的抗癌作用。通过将其与YAP1抑制剂CIL56或谷氨酰胺酶抑制剂CB-839组合,可以在体内和体外放大DFMO的治疗功效。这强调了靶向YAP1介导的谷氨酰胺代谢途径以增强DFMO功效的显著潜力。
结论:我们的发现阐明了YAP1介导的谷氨酰胺代谢是对抗DFMO的关键旁路机制,在抑制多胺代谢之后。我们的研究为DFMO在转移性和复发性骨肉瘤的“一两次冲击”治疗中的潜在作用提供了有价值的见解。
OBJECTIVE: Osteosarcoma, a highly malignant primary bone tumor primarily affecting adolescents, frequently develops resistance to initial chemotherapy, leading to metastasis and limited treatment options. Our study aims to uncover novel therapeutic targets for metastatic and recurrent osteosarcoma.
METHODS: In this study, we proved the potential of modulating the YAP1-regulated glutamine metabolic pathway to augment the response of OS to DFMO. We initially employed single-cell transcriptomic data to gauge the activation level of polyamine metabolism in MTAP-deleted OS patients. This was further substantiated by transcriptome sequencing data from recurrent and non-recurrent patient tissues, confirming the activation of polyamine metabolism in progressive OS. Through high-throughput drug screening, we pinpointed CIL56, a YAP1 inhibitor, as a promising candidate for a combined therapeutic strategy with DFMO. In vivo, we utilized PDX and CDX models to validate the therapeutic efficacy of this drug combination. In vitro, we conducted western blot analysis, qPCR analysis, immunofluorescence staining, and PuMA experiments to monitor alterations in molecular expression, distribution, and tumor metastasis capability. We employed CCK-8 and colony formation assays to assess the proliferative capacity of cells in the experimental group. We used flow cytometry and reactive oxygen probes to observe changes in ROS and glutamine metabolism within the cells. Finally, we applied RNA-seq in tandem with metabolomics to identify metabolic alterations in OS cells treated with a DFMO and CIL56 combination. This enabled us to intervene and validate the role of the YAP1-mediated glutamine metabolic pathway in DFMO resistance.
RESULTS: Through single-cell RNA-seq data analysis, we pinpointed a subset of late-stage OS cells with significantly upregulated polyamine metabolism. This upregulation was further substantiated by transcriptomic profiling of recurrent and non-recurrent OS tissues. High-throughput drug screening revealed a promising combination strategy involving DFMO and CIL56. DFMO treatment curbs the phosphorylation of YAP1 protein in OS cells, promoting nuclear entry and initiating the YAP1-mediated glutamine metabolic pathway. This reduces intracellular ROS levels, countering DFMO\'s anticancer effect. The therapeutic efficacy of DFMO can be amplified both in vivo and in vitro by combining it with the YAP1 inhibitor CIL56 or the glutaminase inhibitor CB-839. This underscores the significant potential of targeting the YAP1-mediated glutamine metabolic pathway to enhance efficacy of DFMO.
CONCLUSIONS: Our findings elucidate YAP1-mediated glutamine metabolism as a crucial bypass mechanism against DFMO, following the inhibition of polyamine metabolism. Our study provides valuable insights into the potential role of DFMO in an \"One-two Punch\" therapy of metastatic and recurrent osteosarcoma.