Selective targeting of different populations of myeloid-derived suppressor cells by histone deacetylase inhibitors

Abstract

Myeloid-derived suppressor cells (MDSCs) are widely implicated in negative regulation of immune responses in cancer. Inhibition of class I histone deacetylases (HDAC) with entinostat has anti-MDSC activity. However, as single agent, it did not delay tumor growth in EL4 and LLC tumor models. Here, we found that entinostat reduced immune suppressive activity of only one type of MDSC-polymorphonuclear, PMN-MDSC, whereas it had no effect on monocytic M-MDSC or macrophages. M-MDSC had high amount of class II HDAC-HDAC6, which was further increased after the treatment of mice with entinostat. Inhibition of HDAC6 with ricolinostat reduced suppressive activity of M-MDSC, but did not affect PMN-MDSC or delayed tumor growth. However, combination of entinostat and ricolinostat abrogated suppressive activity of both populations of MDSC and substantially delayed tumor progression. Thus, inactivation of MDSC required targeting of both major subsets of these cells via inhibitors of class I and class II HDAC.

Keywords: Antitumor response; Entinostat; Histone deacetylase; Macrophages; Myeloid-derived suppressor cells; Ricolinostat.

Fig. 1

Entinostat did not show anti-tumor activity but affected the presence of myeloid cell. a, b EL4 tumor-bearing mice were treated with vehicle or entinostat (10 mg/kg) daily on days 7—17. a Kinetics of tumor progression (n = 10). b The presence of indicated cell populations in spleens and tumors measured on day 17 by flow cytometry. N = 5. P values were calculated in two-tailed Student’s t-test. Results are shown as mean ± SD. c, d, LLC tumor-bearing mice were treated with vehicle or entinostat daily on days 11—28. c Kinetics of tumor progression (n = 5). d The presence of indicated cell populations in spleens and tumors measured on day 28 by flow cytometry. N = 11 for vehicle-treated group, n = 13 for entinostat-treated group. P values were calculated in two-tailed Student’s t-test. Results are shown as mean ± SD. PMN-MDSC: CD45⁺CD11b⁺Ly6G⁺Ly6C^low, M-MDSC: CD45⁺CD11b⁺Ly6G⁻Ly6C^high, macrophage: CD45⁺Ly6C⁻F4/80⁺Ly6G⁻

Fig. 2

Entinostat impairs the immunosuppressive capacity of PMN-MDSC but not of M-MDSC. PMN-MDSC or M-MDSC was purified from spleens and tumors of EL4 tumor-bearing mice (a, b) or LLC tumor-bearing mice (c), which were treated with vehicle or entinostat (10 mg/kg) for 2 weeks. MDSCs were cocultured with PMEL splenocytes and gp100 peptide for 2 days. T cell proliferation was measured in triplicate by ³H-thymidine uptake. T cell proliferation in the absence of MDSC was set as 100%, and percent of change in each experiment was calculated. N = 5–11 in different experiment (shown on each panel). P values were calculated in two-tailed Student’s t-test. Results are shown as mean ± SD. *p < 0.05. d RNA was extracted from MDSC from spleens of EL4 tumor-bearing mice, and expression of indicated mRNA was analyzed by qRT-PCR (n = 4–5). P values were calculated in two-tailed Student’s t-test. Results are shown as mean ± SEM

Fig. 3

HDAC expression in MDSC. a Total RNA was extracted from splenic PMN-MDSC and M-MDSC purified from EL4 tumor-bearing mice and analyzed by qRT-PCR (n = 4). P values were calculated in two-tailed Student’s t-test. b HDAC protein expression in whole cell lysate of splenic PMN-MDSC and M-MDSC from EL4 tumor-bearing mice (n = 2) was analyzed by western blot. c Class I HDAC activity was measured in PMN-MDSC and M-MDSC from spleen of EL4 tumor bearing mice treated with vehicle or entinostat (10 mg/kg) for 2 weeks (n = 5). P values were calculated in two-tailed Student’s t-test. dHDAC6 expression in splenic PMN-MDSC and M-MDSC from EL4 tumor-bearing mice treated with vehicle or entinostat for 2 weeks was analyzed by qRT-PCR (n = 4–5). P values were calculated in two-tailed Student’s t-test

Fig. 4

Anti-tumor effect of HDAC inhibitors. a EL4 tumor-bearing mice were treated with entinostat in combination with anti-CSF1R antibody (30 mg/kg, twice a week). N = 4. P values were calculated in two-way Anova test. Results are shown as mean ± SEM. b, c. EL4 (n = 9) or LLC (n = 5) tumor-bearing mice were treated with entinostat in combination with ricolinostat. Tumor size was measured. Results are shown as mean ± SEM. P values were calculated in two-way Anova test. d M-MDSC and PMN-MDSC were purified from the spleens of EL4 tumor-bearing mice treated with entinostat and ricolinostat and cocultured with PMEL splenocytes and gp100 peptide. T cell proliferation was measured by ³H-thymidine uptake. N = 3–8 shown on each plot. P values were calculated in two-tailed Student’s t-test. Results are shown as mean ± SD