Immunomodulation of HDAC Inhibitor Entinostat Potentiates the Anticancer Effects of Radiation and PD-1 Blockade in the Murine Lewis Lung Carcinoma Model

Abstract

Although the combination of radiotherapy and immunotherapy has proven to be effective in lung cancer treatment, it may not be sufficient to fully activate the antitumor immune response. Here, we investigated whether entinostat, a histone deacetylase inhibitor, could improve the efficacy of radiotherapy and anti-PD-1 in a murine syngeneic LL/2 tumor model. A total of 12 Gy of X-rays administered in two fractions significantly delayed tumor growth in mice, which was further enhanced by oral entinostat administration. Flow cytometry-aided immune cell profiling revealed that entinostat increased radiation-induced infiltration of myeloid-derived suppressor cells and CD8⁺ T cells with decreased regulatory T-cells (Tregs). Transcriptomics-based immune phenotype prediction showed that entinostat potentiated radiation-activated pathways, such as JAK/STAT3/interferon-gamma (IFN-γ) and PD-1/PD-L1 signaling. Entinostat augmented the antitumor efficacy of radiation and anti-PD-1, which may be related to an increase in IFN-γ-producing CD8⁺ T-cells with a decrease in Treg cells. Comparative transcriptomic profiling predicted that entinostat increased the number of dendritic cells, B cells, and T cells in tumors treated with radiation and anti-PD-1 by inducing MHC-II genes. In conclusion, our findings provided insights into how entinostat improves the efficacy of ionizing radiation plus anti-PD-1 therapy and offered clues for developing new strategies for clinical trials.

Keywords: anti-PD-1; antitumor immunity; entinostat; lung cancer; radiation.

Figure 1

Entinostat increases radiation-induced tumor growth delay in a Lewis lung cancer model. (A) Scheme for radiation and entinostat treatments. (B) Tumor growth curves showing reduced tumor growth in LL/2 tumor-bearing C57/BL6 mice treated with entinostat and/or IR. (C) Comparison of tumor weight at day 15 after irradiation. (D) Body weight changes of LL/2 tumor-bearing mice after treatments. (E) Survival curves of LL/2 tumor-bearing mice after entinostat and/or IR treatment. Data are mean ± S.D. (n = 8). * p < 0.05; ** p < 0.01; *** p < 0.001. Mice were categorized as dead when the tumor volume reached 2000 mm³.

Figure 2

Entinostat modulates radiation-induced MDSC infiltration in tumors. (A) Gating strategy for the evaluation of the myeloid-derived suppressor cells (MDSC) population using flow cytometry. Representative density plots are shown. (B) Flow cytometric analysis of MDSC populations. Radiation increased the infiltration of total MDSCs in tumors. Entinostat further enhanced PMN-MDSC but not M-MDSC. PD-L1 expression on MDSCs was further increased by IR and entinostat combination. Data are mean ± S.D. (n ≥ 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Entinostat increases radiation-induced CD8⁺ T cell infiltration in tumors. (A) Gating strategy for the evaluation of the T cell population using flow cytometry. Representative density plots are shown. (B) Flow cytometric analysis of T cell populations. Entinostat further enhanced radiation-increased infiltration of IFN-γ-producing CD8⁺ T cells in tumors. The combination of entinostat and IR decreased Treg cells in tumors. Data are mean ± S.D. (n ≥ 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Transcriptomic analysis reveals entinostat-mediated immune modulation in the tumor microenvironment. (A) Heatmap representing hierarchical clustering of all DEGs in four groups. (B) ImmuCellAI-mouse based estimation of immune cells infiltrating in tumors. (C) Comparison of GSVA scores of IL6-JAK-STAT3, mTORC1, and IFN-γ signaling pathways among three groups. (D) Comparison of expression levels of PD-1 pathway genes. (E) Entinostat induces PD-L1 in LL/2 cells. Dose-dependent induction of PD-L1 in LL/2 cell, which was assessed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Entinostat sensitizes LL/2 tumors to IR plus anti-PD-1 treatment. (A) Scheme for radiation, entinostat, and anti-PD-1 treatments. (B) Tumor growth curves showing delayed growth of LL/2 tumors in mice treated with triple combinations. (C) Comparison of tumor weight at day 15 after irradiation. (D) Photographs of tumors harvested from the mice at day 15 after irradiation. (E–G) Flow cytometric analysis of CD8⁺ T (E), IFN-γ-producing CD8⁺ (F), and Treg (G) cells infiltrated in tumors. Representative density plots are shown. Data are mean ± S.D. (n ≥ 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Entinostat potentiates the combination of IR and anti-PD-1 via upregulating IFN-γ and MHC-II pathways (A) Comparison of estimated infiltration of different immune cell populations in tumors between IR plus anti-PD-1 group and triple combination group. (B) Box plots showing the difference in each immune cell population between the two groups. (C) Dot plots showing enrichment of DEGs related to interferon pathways. (D) Dot plots showing enrichment of DEGs related to MHC genes between two groups. (E) Higher expression levels of MHC-II pathway genes in the triple combination group than those in the IR plus anti-PD-1 group. (F) Entinostat induces MHC-II in LL/2 cells. Dose-dependent induction of MHC-II in LL/2 cells, which was assessed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001.