Research ArticleCancer

MEK inhibition enhances oncolytic virus immunotherapy through increased tumor cell killing and T cell activation

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Science Translational Medicine  12 Dec 2018:
Vol. 10, Issue 471, eaau0417
DOI: 10.1126/scitranslmed.aau0417
  • Fig. 1 MEK inhibition augments T-VEC–mediated cell lysis in vitro and increases viral replication.

    Cell viability determined by MTS assay. (A to D) Left: Cells were treated with either T-VEC alone or trametinib (MEKi) or a combination of T-VEC and MEKi . Right: HSV-1 titers as measured by plaque assay from cells treated with either T-VEC alone (blue bar) or T-VEC and MEKi (purple bar). Only significant differences are indicated. PFU, plaque-forming units. (E) Western blot of cell lysate collected at 24 hours after mT-VEC (0.1 MOI) infection of SK-MEL-28, mock infected, MEKi (10 nM), or combination treatment. (F) Infection metric analysis by LumaCyte (left panel) of SK-MEL-28 cells (mock), treated with 10 nM trametinib (MEKi), 1 MOI of T-VEC, or trametinib and T-VEC. The right panel shows a time course for untreated cells (dotted black line), or those treated with 0.1 MOI of T-VEC (dotted blue line) or 1 MOI of T-VEC (solid blue line). (G) PCA of the infection metric. Each experiment was performed in triplicate and is conducted at least twice with similar results. Data are presented as means ± SEM, and statistical differences between groups were measured by using two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 MEK inhibition enhances T-VEC–induced inhibition of human melanoma xenograft growth in vivo and promotes tumor cell apoptosis.

    (A) NSG (nonobese diabetic/severe combined immunodeficient interleukin-2 receptor gamma chain null) mice (n = 5 per group) were implanted subcutaneously with human melanoma SK-MEL-28 cells (8 × 106) on day 0 (d0), treated via intratumoral injection with sterile water or T-VEC (1 × 105 PFU) on days 35, 40, and 45, and MEKi (trametinib; 0.5 mg/kg) or vehicle [0.2% Tween 80 and 0.5% hydroxypropyl methyl cellulose (HPMC)] was given from days 35 to 43 via oral gavage. Red arrows indicate days when T-VEC was injected, and the blue bar on top indicates days of trametinib (MEKi) treatment. (B) Mean tumor area. (C) Representative images obtained from immunohistochemical staining of tumors for Ki67 at day 36, (D) HSV-1 gD, (E) pERK1/2, and (F) cleaved caspase 3. Right panels indicate quantification of positive cells. Scale bars are as indicated. Each experiment was repeated at least twice with similar results. Data are presented as means ± SEM, and statistical differences between groups were measured by using one-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Only significant differences are indicated.

  • Fig. 3 Combination of T-VEC and MEK inhibition enhances tumor regression in an immune-competent murine melanoma model, promotes recruitment of CD8+ T cells, and establishes long-term memory.

    (A) Treatment schema: Red arrows indicate days of mT-VEC treatment, and the blue bar on top indicates trametinib (MEKi) treatment. (B) Mean tumor area of mice from treated groups at day 45. (C) Survival of mice. (D) Rechallenge of mice cured in (C). (E) Flow cytometry analysis of tumors at day 24. Bar graphs (n = 6) indicating the percentage of positive CD8 T cells, CD8+IFN-γ, CD8+granzyme B, and CD8+Ki67 T cells, respectively. (F) Immunohistochemical staining of CD8+ T cells in the tumor. Scale bar is as indicated. (G) Quantification of CD8+ cells. (H) Bar graph indicating CD4+ and CD4+FoxP3 (Tregs) and ratio of CD8+ T cells to Tregs. Each experiment was conducted at least twice with similar results. Data are presented as means ± SEM, and statistical differences between groups were measured by using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Only significant differences are indicated.

  • Fig. 4 Depletion of CD8+ T cells abrogates the effects of T-VEC and MEKi combination therapy.

    (A) C57BL/6J mice (n = 5 per group) were implanted with D4M3A murine melanoma cells, and mice were treated as described in Materials and Methods. Red arrows indicate days of mT-VEC treatment, the blue bar on top indicates days of trametinib (MEKi) treatment, and black arrows indicate days where depletion antibodies against CD4, CD8, and clodronate were injected. (B) Mean tumor area of mice treated from different groups as indicated. (C) Survival of mice. (D and E) Flow cytometric analysis of tumor-infiltrating T cells on day 24. Bar graphs show the percentage of (D) CD45+CD3+CD4+ and (E) CD45+CD3+CD8+ cells. Each experiment was repeated at least twice with similar results. Data are presented as means ± SEM, and statistical differences between groups were measured by using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Only significant differences are indicated.

  • Fig. 5 Combination of T-VEC and MEK inhibition induces viral-specific CD8+ T cells and increases melanoma antigen–specific CD8+ T cell responses.

    C57BL/6J mice implanted subcutaneously in the right flank with 3 × 105 D4M3A cells and treated with mT-VEC (1 × 106 PFU) or sterile water intratumorally for three doses on days 15, 19, and 22 and/or trametinib (0.5 mg/kg) or vehicle (0.2% Tween 80 and 0.5% HPMC) orally once daily on days 15 to 19. Tumors were harvested on day 24, and cells were dissociated and analyzed by flow cytometry. Percentages of live CD45+ cells, CD3+ cells, and CD3+CD8+ subsets from the mock, T-VEC monotherapy, MEKi monotherapy, and T-VEC + MEKi combination groups were analyzed and compared. Tumor-infiltrating CD8+ T cells were analyzed with (A) HSV-1–specific H-2Kb–HSV-1 gB dextramer, (B) melanoma antigen–specific H-2Db–gp100 dextramer, and (C) H-2Kb–TRP2 dextramer. Quantitative analysis is shown in the bar graphs on the right. These experiments were conducted at least twice with similar results. Data are presented as means ± SEM, and statistical differences between groups were measured by using one-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001. Only significant differences are indicated.

  • Fig. 6 Batf3+ DCs play a role in the antitumor activity and antigen spreading associated with combination treatment with T-VEC and MEK inhibition.

    C57BL/6J mice (B6, n = 7) and Batf3−/− mice (n = 7) were implanted with D4M3A murine melanoma cells and either mock treated or treated with T-VEC and trametinib as described in Materials and Methods. (A) Survival of mice. (B) Mean tumor area. (C to F) Flow cytometry analysis of tumors obtained from B6 and Batf3−/− mice on day 24. (C) Bar graph indicating the percentage and number of tumor-infiltrating total CD8+ T cells and the frequency of CD8+IFN-γ+ and CD8+granzyme B+ T cells, respectively. (D) CD8+Ki67+ T cells. (E) CD4+FoxP3+ Tregs. (F) Percentage of HSV 1-gB+, murine gp100+, and TRP2+CD8+ T cells, respectively. These experiments were repeated at least twice with similar results. Data are presented as means ± SEM, and the statistical differences between groups were measured by two-tailed Student’s t test. **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 7 T-VEC and MEK inhibition reprogram immune silent tumors into immune inflamed tumors and induces expression of PD-1 and PD-L1.

    C57BL/6J mice were implanted subcutaneously in the right flank with 3 × 105 D4M3A cells and treated with mT-VEC (1 × 106 PFU) intratumorally for three doses on days 15, 19, and 22 and/or trametinib (0.5 mg/kg) orally once daily on days 15 to 19. Tumors were harvested on day 24, total RNA was isolated, and gene expression analysis was performed using the NanoString PanCancer Immune panel as described in Materials and Methods. (A) An inflammatory 16-gene expression profile was generated from mice (n = 3) treated (as described in Fig. 3D) with mock control (black), trametinib alone (MEKi; blue), mT-VEC alone (red), or a combination of mT-VEC and MEKi (purple). (B) A selected five-gene expression signature represented by genes highly associated with CD8+ T cell activation. (C) Gene expression of PD-1 (right panel) and PD-L1 (left panel). (D) Bar graphs show the mean fluorescence intensity (MFI) of CD45+PD-1+ (left panel) and CD45-PD-L1+ (right panel). Each experiment was performed at least twice with similar results. Data are presented as means ± SEM, and the statistical differences between groups were measured by two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 8 Triple-combination treatment with T-VEC, MEK inhibition, and PD-1 blockade improves therapeutic treatment of melanoma and colon cancer models.

    (A) Treatment schema: Red arrows indicate T-VEC, the blue bar on top indicates trametinib, and brown arrows indicate αPD-1. (B) Mean tumor area. (C) Survival of mice. (D) Rechallenge of mice cured from (B). (E to G) Flow cytometry of tumors at day 24. Bar graph indicating percentage of positive (E) CD45+PD-1+ cells (left panel) and CD8+PD-1+ cells (right panel), (F) CD4+FoxP3+ (left panel) and ratio of effector T cells (Teff) to Tregs (right panel). (G) CD8+ T cells, granzyme B+CD8+ T cells, and Ki67+CD8+ T cells, respectively. (H) Evaluation of triple combination in the CT26 murine colon carcinoma model. Mice were treated as described in Materials and Methods. Each experiment was conducted at least twice with similar results. Data are presented as means ± SEM, and statistical differences between groups were measured by two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/471/eaau0417/DC1

    Methods

    Fig. S1. BRAF inhibitors enhance T-VEC cell killing in BRAF-mutant melanoma cell lines.

    Fig. S2. LumaCyte laser flow cytometry analysis.

    Fig. S3. T-VEC and MEKi–induced apoptosis.

    Fig. S4. Characterization of murine D4M3A cells.

    Fig. S5. Validation of immune cell depletion.

    Fig. S6. Time course analysis of tumor-infiltrating CD8+ T cells during mT-VEC treatment.

    Fig. S7. Analysis of CD8+ T cells from spleen during mT-VEC + MEKi treatment.

    Fig. S8. Characterization of D4M3A tumor cells in Batf3 knockout mice.

    Fig. S9. NanoString gene expression heat maps for all genes profiled and by gene function.

    Fig. S10. MFI expression of PD-1 expression and frequency of PD-1+ cells.

    Fig. S11. Individual tumor growth curves of BALB/c mice bearing CT26 tumors.

    Table S1. Antibodies.

    Table S2. Chemicals.

    Table S3. Commercial assays.

    Table S4. Experimental cell lines.

    Table S5. Experimental models.

    Table S6. Tumor area from mouse studies.

    Data file S1. Gene expression raw data.

  • The PDF file includes:

    • Methods
    • Fig. S1. BRAF inhibitors enhance T-VEC cell killing in BRAF-mutant melanoma cell lines.
    • Fig. S2. LumaCyte laser flow cytometry analysis.
    • Fig. S3. T-VEC and MEKi–induced apoptosis.
    • Fig. S4. Characterization of murine D4M3A cells.
    • Fig. S5. Validation of immune cell depletion.
    • Fig. S6. Time course analysis of tumor-infiltrating CD8+ T cells during mT-VEC treatment.
    • Fig. S7. Analysis of CD8+ T cells from spleen during mT-VEC + MEKi treatment.
    • Fig. S8. Characterization of D4M3A tumor cells in Batf3 knockout mice.
    • Fig. S9. NanoString gene expression heat maps for all genes profiled and by gene function.
    • Fig. S10. MFI expression of PD-1 expression and frequency of PD-1+ cells.
    • Fig. S11. Individual tumor growth curves of BALB/c mice bearing CT26 tumors.
    • Table S1. Antibodies.
    • Table S2. Chemicals.
    • Table S3. Commercial assays.
    • Table S4. Experimental cell lines.
    • Table S5. Experimental models.
    • Legend for table S6

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S6 (Microsoft Excel format). Tumor area from mouse studies.
    • Data file S1 (.txt format). Gene expression raw data.

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