Myelomodulatory treatments augment the therapeutic benefit of oncolytic viroimmunotherapy in murine models of malignant peripheral nerve sheath tumors

Abstract

Introduction: Malignant peripheral nerve sheath tumors (MPNST) pose a significant therapeutic challenge due to high recurrence rates after surgical resection and a largely ineffective response to traditional chemotherapy. An alternative treatment strategy is oncolytic viroimmunotherapy, which can elicit a durable and systemic antitumor immune response and is Food and Drug Administration (FDA)-approved for the treatment of melanoma. Unfortunately, only a subset of patients responds completely, underscoring the need to address barriers hindering viroimmunotherapy effectiveness.

Methods: Here we investigated the therapeutic utility of targeting key components of the MPNST immunosuppressive microenvironment to enhance viroimmunotherapy's antitumor efficacy in three murine models, one of which showed more immunogenic characteristics than the others.

Results: Myelomodulatory therapy with pexidartinib, a small molecule inhibitor of CSF1R tyrosine kinase, and the oncolytic herpes simplex virus T-VEC exhibited the most significant increase in median survival time in the highly immunogenic model. Additionally, targeting myeloid cells with the myelomodulatory therapy trabectedin, a small molecule activator of caspase-8 dependent apoptosis, augmented the survival benefit of T-VEC in a less immunogenic MPNST model. However, tumor regressions or shrinkages were not observed. Depletion experiments confirmed that the enhanced survival benefit relied on a T cell response. Furthermore, flow cytometry analysis following combination viroimmunotherapy revealed decreased M2 macrophages and myeloid-derived suppressor cells and increased tumor-specific gp70+ CD8 T cells within the tumor microenvironment.

Discussion: In summary, our findings provide compelling evidence for the potential to leverage viroimmunotherapy with myeloid cell targeting against MPNST and warrant further investigation.

Keywords: T-VEC; immunotherapy; macrophage targeting; malignant peripheral nerve sheath tumors; oncolytic virotherapy; pexidartinib; trabectedin; tumor microenvironment.

Figure 1

The experimental design used in this report. We first compared different oHSV constructs for their ability to kill MPNST cells and shrink MPNST syngeneic tumors. As none of the viruses was consistently superior to the others, we chose the FDA-approved virus, T-VEC, to further study in combination with drugs postulated to enable viroimmunotherapy. Because in other studies we found trabectedin to be toxic to C57Bl/6 animals, we pivoted to an MPNST model in the Balb/c background to test that combination. Figure created in BioRender.

Figure 2

Comparison of three clinically relevant oncolytic herpes simplex viruses against MPNST. (A) Flow cytometry histograms of MHC Class I (H2-K^b and H2-D^b) and MHC Class II in two murine MPNST cell lines, 67C-4 and #5NPCIS, at baseline and following exposure to murine Interferon-γ. Interferon-γ enhances the expression of H-2K^b in both cell lines. (B) MTS Viability Assay of MPNST cell lines following treatment with oncolytic herpes virus HSV1716 for 96 hours at varying multiplicity of infection (MOI). Samples were run in triplicate. Error bars represent SEM. (C) Dose scheduling of mice implanted subcutaneously with #5NPCIS or 67C-4 (top row). I.Tu., Intratumoral Injection; oHSV, oncolytic herpes simplex virus. Flow analysis of single-cell suspensions obtained from tumors treated with HSV1716 or vehicle control at day seven after the first treatment (bottom row). HSV1716 increases the infiltration of total T cells and CD8⁺ T cells by day 7 in both cell lines (*p≤0.05 and ****p≤0.0001). (D) Average tumor volume (left column) and Kaplan-Meier survival curve (right column) of #5NPCIS (top row) and 67C-4 (bottom row) treated with three clinically relevant oncolytic viruses at the dosing regimen same as panel. A modest but statistically significant impact was observed on overall survival with HSV1716 and C134 in the #5NPCIS model and C134 in the 67C-4 model. Error Bars represent SEM. Statistical significance was assessed using the log-rank test (n = 9-11 per group) (*p≤0.05 and **p≤0.01). (E) In vitro virus replication assays (top row) quantifying the amount of virus produced in 67C-4 and #5NPCIS cells after infection with HSV1716, C134 or T-VEC through plaque assays at an MOI of 0.5 plaque-forming units per cancer cell (n = 4 per group). T-VEC appears to have the highest permissivity. In vivo virus replication assays (bottom row) in mice bearing 67C-4 or #5NPCIS tumors treated with a single intratumoral 1 X 10⁸ pfu dose of virus (n = 4 per group). Error bars represent standard deviation. T-VEC had greater persistence than HSV1716 and C134 in the 67C-4 model.

Figure 3

Screening of combination immunotherapy regimens that target three key players of the immunosuppressive microenvironment in MPNST to leverage virotherapy. Mice treated with the combination of T-VEC and pexidartinib had the greatest increase in median survival compared to other combinations. (A) Schematic of the treatment regimen of mice bearing subcutaneous 67C-4 or #5NPCIS tumors. I.Tu., Intratumoral Injection; oHSV, oncolytic herpes simplex virus. (B) Individual tumor volumes (left column) and Kaplan-Meier survival curve (right column) of #5NPCIS (top row of each combination) and 67C-4 (bottom row of each combination) following combination therapy. The bottommost row shows the combination of T-VEC and pexidartinib in the 67C-4 model. Combining T-VEC with either anti-PD-1 + A8301 or pexidartinib significantly prolongs survival in the 67C-4 tumor model. Among the tested combination regimens, no survival benefit was observed in the #5NPCIS tumor model. The statistical significance of survival data was assessed using the log-rank test (n = 4 to 5 for experimental groups including #5NPCIS mice; n = 7 to 8 for experimental groups including 67C-4 mice) (*p≤0.05 and **p≤0.01).

Figure 4

67C-4 has significant immune cell infiltration, higher tumor mutation burden, and greater expression of a known tumor-associated endogenous retrovirus antigen, envelope glycoprotein gp70, at baseline than #5NPCIS and SN4-4. (A) Deconvolution of RNA-sequencing data via CIBERSORTx to predict immune cell abundance in immunocompetent murine models. (B) Tumor Mutation Burden (C) gp70 expression (D) Immune Checkpoint expression (n=1-2 per tumor model).

Figure 5

Trabectedin augments T-VEC virotherapy in a weakly immunogenic murine model of MPNST through a T cell response contributed by both CD4 and CD8 T cells. (A) Dose scheduling of mice implanted subcutaneously with SN4-4 tumors and treated with T-VEC, Trabectedin, or a combination of T-VEC and Trabectedin. I.Tu., Intratumoral Injection; oHSV, oncolytic herpes simplex virus.; i.v., Intravenous Injection (B) Individual tumor volumes of mice in each treatment group plotted against tumor volumes of control mice (black lines) (n=9 per group). (C) Kaplan Meier survival curves demonstrating prolonged survival in the combination group over monotherapies with T-VEC or trabectedin (n=9 per group) (*p≤0.05, ***p≤0.001 and ****p≤0.0001). (D) Schematic of the treatment regimen of mice bearing SN4-4 tumors undergoing combination therapy and administered with CD4 or CD8 T cell depleting or isotype control antibodies intraperitoneally twice a week (500 ug antibody per injection). (E) Individual tumor volumes of mice in each treatment group plotted against the tumor volumes of control mice undergoing combination therapy and treatment with isotype control antibodies (n = 7-10 per group). (F) Kaplan Meier survival curve demonstrating a decline in survival following treatment with CD4 and CD8 T cell depleting antibodies. The statistical significance of survival data was assessed using the log-rank test (n = 7-10 per group) (**p≤0.01 and ***p≤0.001).

Figure 6

Combination of T-VEC and Trabectedin reduces the proportion of macrophages and myeloid-derived suppressor cells along with an increase in M1/M2 ratio similar to T-VEC. The proportion of NK cells increased significantly in the combination group over monotherapies. Schematic of the treatment regimen of BALB/c mice bearing subcutaneous SN4-4 tumors. The mice received a three intratumoral dose of T-VEC or an equivalent volume of PBS (Vehicle) on day 0, 2 and 4 (n=3-5 per group). Mice were sacrificed at Day 9 to harvest tumors for flow cytometry. Single-cell suspensions were obtained from the tumors, stained, and analyzed for macrophages, myeloid-derived suppressor cells and NK cells. G-MDSCs- Granulocytic-MDSCs; M-MDSCs-Monocytic-MDSCs. The statistical significance was assessed through a one-way ANOVA with Tukey-adjusted post hoc tests (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001).

Figure 7

Combination of T-VEC and Trabectedin significantly increases the proportion of CD8 T cells specific to of a known tumor-associated endogenous retrovirus antigen, envelope glycoprotein gp70 compared to monotherapies with T-VEC or Trabectedin. CD8 T cells were significantly enhanced in the combination group, along with an increase in PD-L1 expression, while retaining the frequency of regulatory T cells similar to T-VEC treated groups. Single-cell populations were obtained from the same experiment described in Figure 6 and stained for a panel of T cell markers (n=3-5 per group). The statistical analysis was performed through a one-way ANOVA with Tukey-adjusted post hoc tests (*p≤0.05 and **p≤0.01).