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

项目总结/摘要 骨肉瘤和尤因肉瘤是最常见的儿童骨恶性肿瘤和努力治疗 尽管进行了几十年的研究，晚期疾病患者的情况仍然令人沮丧。为了解决这一不足，我们 寻求利用溶瘤病毒疗法作为针对肿瘤的双重攻击：直接肿瘤细胞裂解和抗肿瘤 免疫刺激然而，以前的试验溶瘤单纯疱疹病毒（oHSV）在小儿肉瘤 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。提高 为了有效，我们将oHSV与trabectedin联合使用，trabectedin是一种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的 预期，诱导完全消退，并在尤文肉瘤的多种模型中增加生存期， 骨肉瘤这种协同作用的机制仍然是未知的，理解它是优化的关键。 在人体试验中的治疗方法。在我们初步的单细胞RNA测序数据中， 协同作用，我们在联合治疗的肿瘤中观察到比oHSV治疗的肿瘤中多得多的病毒转录物， 与内在抗病毒反应相关的基因表达减少，表明trabectedin 选择性地使肿瘤细胞对感染敏感。我们还发现oHSV诱导TRAILR 2死亡增加， 肿瘤细胞中TRAIL受体的表达和NK细胞中TRAIL表达的增加，指出了第二种可能性， 通过细胞毒性信号传导的协同作用机制。基于这些数据，我们假设， 通过包括肿瘤内源性转录抑制的机制增强oHSV的功效 通过TRAIL-TRAILR 2信号传导的抗病毒应答和增强的NK介导的细胞毒性。首先，我们将 研究降低的内在抗病毒反应和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，包括质谱细胞术（CyTOF）、蛋白质印迹和多色分析。 免疫荧光为了观察相关的肿瘤细胞对oHSV的敏感性，我们将构建一个发光的 oHSV并实时监控其传播。其次，我们将分析TRAIL，TRAILR 2，TRAILR 2抑制剂， 使用CyTOF、蛋白质印迹、ELISA、多色免疫荧光和qPCR的胱天蛋白酶-8途径表达， 与oHSV治疗的肿瘤相比，联合治疗的肿瘤中的细胞凋亡率。为了确定TRAILR 2信号传导是否是 为了达到联合疗效，我们将使用CRISPR-Cas9敲除肿瘤细胞TRAILR 2，并比较 在野生型和敲除模型中的单一和组合治疗功效。最终，我们将阐明 协同作用的机制，通过双管齐下的分析，有可能揭示许多可推广的 对骨肉瘤和尤文肉瘤的临床应用和发展前景进行了展望 针对小儿肉瘤的协同治疗。

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.