Epigenetic therapy activates type I interferon signaling in murine ovarian cancer to reduce immunosuppression and tumor burden

Abstract

Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45⁺ immune cells and the percentage of active CD8⁺ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

Keywords: 5-azacytidine; histone deacetylase inhibitors; immunosuppression; ovarian cancer; type I interferon.

Fig. 1.

Pretreatment of tumor epithelial cells with AZA and transplantation into untreated C57BL/6 mice lead to decreased tumor-associated ascites and increased overall survival. (A) Treatment schematic for in vitro treatment of cultured ID8-VEGF-Defensin cells (AZA, 500 nM; MS275, 30 or 100 nM; ITF, 100 nM). A, AZA; ITF, givinostat; MS, entinostat. (B and C) Ascites volume drained from mice 4 to 5 wk after pretreated tumor injection. Mean ± SEM is shown. A10, MS3, MS10: n = 7 to 30 mice, three biological replicates; A3-10: n = 9 mice, two biological replicates; MS17, ITF17, A17, A+MS17, and A+ITF17: n = 9 or 10 mice, one biological replicate. Statistical outliers were removed using Peirce’s criterion, and significance was determined by Mann–Whitney t test. (D) Survival of mice in days, with median survival shown. Significance was determined using a log-rank (Mantel–Cox) test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Pretreatment of tumor epithelial cells with AZA and an HDACi leads to alterations in the numbers and activation of immune cell populations in tumor-associated ascites. ID8-VEGF-Defensin cells were pretreated (AZA, 500 nM; entinostat, 30 or 100 nM; givinostat, 100 nM) and injected into mice. Cells were analyzed from the drained ascites fluid (Fig. 1 A–C). (A) Immune cells per mL separated via Percoll gradient (n = 6 to 12 mice, two or three biological replicates). (B) CD45⁺ cells per mL identified via Percoll gradient and FACS (n = 6 to 11 mice, two biological replicates). Mean ± SEM is shown, and significances were determined by Mann–Whitney t test. (C–J) All cells from ascites were analyzed via FACS (n = 5 to 9 mice, one biological replicate). Mean ± SEM is shown, and significances were determined by one-way ANOVA. (C) CD45⁺ cells per mL of ascites. (D–J) Response of immune cell subpopulations to tumor cells pretreated ex vivo with AZA (A10). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Addition of immune checkpoint inhibition to epigenetic therapy in an intact mouse model decreases tumor burden and increases survival. (A) In vivo treatment schematic of AZA (0.5 mg/kg), entinostat (2 mg/kg), givinostat (2 mg/kg), and α-PD-1 (200 μg per mouse). (B) Volume of ascites fluid drained at week 6. Mean ± SEM is shown and significances were determined by one-way ANOVA. All significances are compared with mock; **P < 0.01, ***P < 0.001; ns, not significant; n = 8 to 10 mice per group. (C–F) Survival of the mice in days, with median survival shown. Significances were determined by log-rank (Mantel–Cox) test; n = 10 mice per group.

Fig. 4.

Epigenetic therapy and α-PD-1 increase the number and activation of immune cells in the tumor microenvironment. Mice were treated as described in Fig. 3A. Cells from ascites fluid drained at week 6.5 were analyzed via FACS. Median, 25th and 75th percentiles, and range are plotted for each experimental arm, and significances were determined by Mann–Whitney t test. Significances compared with mock are marked with *, and significances compared with AZA are marked with #. *^/#P < 0.05, **^/##P < 0.01, ***^/###P < 0.001. (A) % T effector cells (CD8⁺IFN-γ⁺) of T cells. (B) % T helper cells (CD4⁺IFN-γ⁺) of T cells. (C) % activated NK cells (NK1.1⁺, IFN-γ⁺) of NK1.1⁺ cells. (D) % myeloid-derived suppressor cells (GR-1⁺, CD11b⁺, F4/80⁻, MHCII⁻) of CD45⁺ cells. (E) % macrophages (CD11b⁺, F4/80⁺) of CD45⁺ cells. (F) % CD3⁺ T cells of CD45⁺ cells. (A–C and F) n = 4 to 9 mice per group. (D and E) n = 2 to 9 mice per group.

Fig. 5.

Blockade of IFNAR1 inhibits the actions of AZA. (A) Treatment schematic for the mice. Mice were treated with AZA (0.5 mg/kg) or saline as described in Fig. 3. Anti-IFNAR1 was injected i.p. (0.5 mg per mouse) every 3 d, beginning 1 d before the AZA regimen. (B) Volume of ascites drained from the mice at week 4.5. Mean ± SEM is shown, and significances were determined by Mann–Whitney t test; n = 8 to 10 mice per group. (C) Survival of the mice in days, with median survival shown. Significances were determined by log-rank (Mantel–Cox) test; n = 10 mice per group. (D–F) Median, 25th and 75th percentiles, and range are plotted, and significances were determined by Mann–Whitney t test; n = 6 to 9 mice per group. (D) CD45⁺ cells per mL of ascites. (E) % T effector cells (CD8⁺IFNγ⁺) of CD3⁺ T cells. (F) % activated NK cells (NK1.1⁺, IFNγ⁺) of NK1.1⁺ cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

AZA+HDACi combination therapy is less effective at reducing tumor burden and increasing survival in an immunodeficient mouse model. (A) Treatment schematic for in vivo treatment of NSG mice with AZA and HDACis entinostat or givinostat. (B) Fold change in ascites volume drained at week 5.5 (NSG) or 6 (C57BL/6). The C57BL/6 data from Fig. 3B are shown here for direct comparison; n = 3 to 10 mice per group. (C) NSG mouse survival in days, with median survival shown. Significances were determined by a log-rank (Mantel–Cox) test; n = 10 mice per group. (D) Ascites volume drained at week 4.5 or 4 from C57BL/6 or NSG mice, respectively, treated with AZA and anti-IFNAR1 as shown in Fig. 5 A and B; n = 8 to 10 mice per group. (E) % dead, CD45⁻, nonimmune ascites cells (Live/Dead stain⁺, CD45⁻) from NSG ascites fluid; n = 5 to 10 mice per group. (B, D, and E) Mean ± SEM is shown, and significances were determined by Mann–Whitney t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

Ex vivo treatment of ID8-VEGF-Defensin cells with low-dose AZA decreases viable cell number, increases apoptosis, and disrupts the cell cycle. (A) Three or 10 d in vitro treatment with 500 nM AZA. (B) Total number of cells relative to mock; n = 3. (C) Quantification of c-PARP levels in AZA-treated cells relative to mock; n = 3. (D) A representative Western blot of c-PARP levels. (E and F) Percentage of annexin V⁺ and 7-AAD⁺ apoptotic cells. Representative flow cytometry data are shown (E) along with quantification (F); n = 3. (G and H) Cell-cycle analysis, determined by BrdU incorporation and 7-AAD staining of DNA content; n = 3. Mean ± SEM is shown, and significances were determined by Mann–Whitney t test. *P < 0.05, **P < 0.01, ***P < 0.001.