(P 107) TRABECTEDIN AUGMENTS ONCOLYTIC HERPES SIMPLEX VIROIMMUNOTHERAPY EFFICACY IN PRECLINCAL BONE SARCOMA MODELS THROUGH ENHANCED IMMUNE-MEDIATED MECHANISMS

Objective: Treatment of patients with advanced cases of osteosarcoma, the most common pediatric bone sarcoma, remains largely unsuccessful. Decades of research yielded little improvement to the treatment regimens for patients affected by this bone sarcoma. We aim to improve patient outcomes through the use of oncolytic herpes simplex viroimmunotherapy, which induces tumor cell-specific lysis and anti-tumor immune activation. After observing the impressive synergy between oncolytic herpes simplex viroimmunotherapy and the FDA-approved drug trabectedin, we sought to determine the mechanisms of this efficacy. Previous results indicated that treatment of immunodeficient human Ewing sarcoma xenograft models may depend on enhanced oncolytic herpes simplex viroimmunotherapy spread through trabectedin-mediated disruption of tumor intrinsic antiviral gene expression. However, due to species-dependent differences, mouse syngeneic models do not display sufficient viral spread for this mechanism of synergy. Thus, based on the known ability of trabectedin to reduce immunosuppressive monocytes and macrophages, we hypothesized that the observed synergy is immune-mediated in immunocompetent osteosarcoma tumor models.

Methods: In multiple preclinical syngeneic mouse models of osteosarcoma, we combined oncolytic herpes simplex viroimmunotherapy with trabectedin to determine the efficacy and survival benefits of the combination therapy with and without immune effector populations. We employed single-cell RNA sequencing and flow cytometry to probe the synergistic mechanism in immunocompetent osteosarcoma models based on differences in immune infiltrate and polarization between treatment groups.

Results: Multiple models of osteosarcoma treated with trabectedin combined with oncolytic herpes simplex viroimmunotherapy showed impressive synergy, with the majority displaying complete or partial tumor regressions. The combination therapy exhibited significant survival benefits (p < 0.05) alongside low average tumor burdens across immunocompetent models. Single-cell RNA sequencing data showed decreased immunosuppressive, M2-like gene expression in monocytes and macrophages in combination-treated tumors compared to those treated with the viroimmunotherapy alone. Flow cytometry also supported a decrease in M2-like macrophages in combination-treated tumors. In correlation with these observations, we detected enhanced granzyme gene expression in NK and CD8 T cells from combination-treated tumors as compared to monotherapy-treated tumors, thereby displaying potential anti-tumor immune activation. Indeed, the depletion of NK cells or CD8 T cells caused a loss of the combination efficacy, indicating that NK or CD8 T cells are necessary for combination efficacy. Interestingly, the depletion of CD4 T cells resulted in increased efficacy compared to the combination control, correlating with single-cell RNA sequencing data that nearly all Cd4+ T cells in the tumor microenvironment are Foxp3+ T regulatory cells.

Conclusion: Our results indicate that trabectedin enhances oncolytic herpes simplex viroimmunotherapy in preclinical immunocompetent osteosarcoma models through immune-mediated anti-tumor mechanisms. Trabectedin removes the immunosuppressive barriers of anti-inflammatory, pro-tumor monocytes and macrophages and frees immune effector cells, namely NK and CD8 T cells, to induce tumor cell death. This immune-mediated mechanism may synergize with our observation of increased viral spread in human tumors to generate greater combinatorial efficacy in patients.