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

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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.