Mechanisms of Synergy between Oncolytic Herpes Simplex Virus and Trabectedin in Pediatric Bone Sarcomas

溶瘤单纯疱疹病毒与曲贝替定治疗小儿骨肉瘤的协同作用机制

• 批准号: 10607503
• 负责人: Emily Ringwalt
• 金额: $ 3.59万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607503
• 关键词: Address; Antiviral Response; Apoptosis; Binding; CASP8 and FADD-like apoptosis regulating protein; CASP8 gene; CRISPR/Cas technology; Cell Death; Cells; Cessation of life; Childhood; Clinical Trials; Color; Combined Modality Therapy; Cytokine Signaling; Cytolysis; Cytometry; DNA Binding Agent; Data; Data Analyses; Development; Ensure; Enzyme-Linked Immunosorbent Assay; Ewings sarcoma; Exhibits; FDA approved; Future; Gene Expression; Genetic Transcription; Human; Immunocompetent; Immunofluorescence Immunologic; Immunologic Deficiency Syndromes; Immunologic Stimulation; Immunosuppression; In complete remission; Infection; Interferons; Knock-out; Label; Lead; Macrophage; Malignant Neoplasms; Mediating; Methods; Modeling; Monitor; Natural Killer Cells; Oncogenic Viruses; Oncolytic; Pathway interactions; Patients; Pattern; Pattern recognition receptor; Pharmaceutical Preparations; Production; Proteomics; Rationalization; Reporter Genes; Reporting; Research; Role; Sampling; Signal Transduction; TLR4 gene; TNFRSF10B gene; TNFSF10 gene; Testing; Therapeutic; Time; Transcript; Treatment Efficacy; Viral; Viral Proteins; Virus; Virus Diseases; Western Blotting; Work; advanced disease; antitumor effect; bone; cancer therapy; cancer type; childhood sarcoma; clinical application; comparative efficacy; cytotoxic; cytotoxicity; expectation; gene product; immunosuppressive macrophages; improved; in vivo; inhibitor; innovation; insight; monocyte; mouse model; neoplastic cell; novel; novel therapeutics; oncolysis; oncolytic herpes simplex virus; oncolytic virotherapy; osteosarcoma; protein expression; receptor expression; response; single-cell RNA sequencing; success; synergism; tumor

PROJECT SUMMARY/ABSTRACT Osteosarcoma and Ewing sarcoma are the most common pediatric bone malignancies and efforts to treat patients with advanced disease remain dismal, despite decades of research. To address this inadequacy, we seek to leverage oncolytic virotherapy as a twofold attack against tumors: direct tumor cell lysis and antitumor immune stimulation. However, previous trials of oncolytic herpes simplex virus (oHSV) in pediatric sarcomas displayed limited responses, likely due to immunosuppressive monocytes and macrophages. To improve efficacy, we combined oHSV with trabectedin, an FDA-approved DNA-binding agent known to mitigate monocytes and macrophages. Strikingly, we found that the efficacy of this combination far surpassed our expectations, inducing complete regressions and increased survival in multiple models of Ewing sarcoma and osteosarcoma. The mechanism for this synergy remains unknown and understanding it is critical to optimize approaches to therapy in human trials. In our preliminary single-cell RNA sequencing data investigating this synergy, we observed far more viral transcripts in combination-treated tumors than in oHSV-treated tumors and a decrease in the expression of genes associated with the intrinsic antiviral response, suggesting that trabectedin selectively sensitizes tumor cells to infection. We also found that oHSV induced increased TRAILR2 death receptor expression in tumor cells and increased TRAIL expression in NK cells, pointing to a second potential mechanism for synergy through cytotoxic signaling. Based on these data, we hypothesize that trabectedin augments oHSV efficacy through mechanisms that include transcriptional inhibition of tumor-intrinsic antiviral responses and enhanced NK-mediated cytotoxicity via TRAIL-TRAILR2 signaling. Firstly, we will investigate the decreased intrinsic antiviral response and the oHSV-induced tumor cell death pathways under combination therapy via proteomic methods, including mass cytometry (CyTOF), western blot, and multi-color immunofluorescence. To observe the related tumor cell sensitivity to oHSV, we will construct a luminescent oHSV and monitor its spread in real-time. Secondly, we will analyze TRAIL, TRAILR2, TRAILR2 inhibitors, and caspase-8 pathway expressions using CyTOF, western blot, ELISA, multi-color immunofluorescence, and qPCR, in combination-treated tumors compared to oHSV-treated tumors. To determine whether TRAILR2 signaling is necessary for combination efficacy, we will use CRISPR-Cas9 to knock out tumor cell TRAILR2 and compare single and combination treatment efficacies in wild-type and knock-out models. Ultimately, we will illuminate mechanisms of synergy through a two-pronged analysis, with the potential to reveal numerous generalizable vulnerabilities in osteosarcoma and Ewing sarcoma for clinical application and the development of future synergistic treatments against pediatric sarcomas.

项目摘要/摘要 骨肉瘤和尤因肉瘤是最常见的儿科骨恶性肿瘤和治疗的努力 尽管进行了数十年的研究，患有晚期疾病的患者仍然令人沮丧。为了解决这一不足，我们 寻求利用癌性病毒疗法作为对肿瘤的双重攻击：直接肿瘤细胞裂解和抗肿瘤 免疫刺激。但是，先前对小儿肉瘤中溶瘤疱疹病毒（OHSV）的试验 显示有限的反应，可能是由于免疫抑制单核细胞和巨噬细胞所致。改进 功效，我们将OHSV与trabectedin合并，这是一种已知可减轻的FDA批准的DNA结合剂 单核细胞和巨噬细胞。令人惊讶的是，我们发现这种组合的功效远远超过了我们 期望，引起完整的回归和增加的ewing肉瘤和生存率 骨肉瘤。这种协同作用的机制仍然未知，并且了解优化至关重要 人类试验中的治疗方法。在我们的初步单细胞RNA测序数据中 协同作用，我们观察到联合治疗的肿瘤中的病毒转录本要比在OHSV处理的肿瘤和 与内在抗病毒反应相关的基因表达的降低，表明trabectedin 有选择地将肿瘤细胞敏感到感染。我们还发现OHSV诱导的Trailr2死亡增加 肿瘤细胞中的受体表达并增加了NK细胞中的Trail表达，指出第二势 通过细胞毒性信号传导协同作用的机制。基于这些数据，我们假设trabectedin 通过包括转录抑制肿瘤内在的机制增强OHSV的功效 抗病毒反应和通过TRAIL-TRAILR2信号传导增强了NK介导的细胞毒性。首先，我们会的 研究内在的抗病毒反应减少和OHSV诱导的肿瘤细胞死亡途径 通过蛋白质组学方法的组合疗法，包括质量细胞术（Cytof），Western印迹和多色 免疫荧光。为了观察相关的肿瘤细胞对OHSV的敏感性，我们将构建发光 OHSV并实时监视其传播。其次，我们将分析TRAIL，TRAILR2，TRAILR2抑制剂，以及 使用Cytof，Western印迹，ELISA，多色免疫荧光和qPCR，Caspase-8途径表达式 与OHSV处理的肿瘤相比，在组合处理的肿瘤中。确定trailr2信号是否是 组合功效所需的必要 野生型和敲除模型的单一和组合治疗效率。最终，我们将阐明 通过两管齐的分析来协同作用的机制，有可能揭示许多可推广的 骨肉瘤和尤因肉瘤的漏洞用于临床应用和未来的发展 针对小儿肉瘤的协同治疗方法。