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. 2024 Jan 4;25(1):663.
doi: 10.3390/ijms25010663.

Combination Therapy with a TLR7 Agonist and a BRD4 Inhibitor Suppresses Tumor Growth via Enhanced Immunomodulation

Yong-Si Liu  1 , Jia-Xin Wang  1 , Guang-Yi Jin  1 , Ming-Hao Hu  1 , Xiao-Dong Wang  1
Affiliations

Combination Therapy with a TLR7 Agonist and a BRD4 Inhibitor Suppresses Tumor Growth via Enhanced Immunomodulation

Yong-Si Liu et al. Int J Mol Sci. .

Abstract

JQ-1 is a typical BRD4 inhibitor with the ability to directly fight tumor cells and evoke antitumor immunity via reducing the expression of PD-L1. However, problems arise with the development of JQ-1 in clinical trials, such as marked lymphoid and hematopoietic toxicity, leading to the investigation of combination therapy. SZU-101 is a TLR7 agonist designed and synthesized by our group with potent immunostimulatory activity. Therefore, we hypothesized that combination therapy of SZU-101 and JQ-1 would target innate immunity and adaptive immunity simultaneously, to achieve a better antitumor efficacy than monotherapy. In this study, the repressive effects of the combination administration on tumor growth and metastasis were demonstrated in both murine breast cancer and melanoma models. In 4T1 tumor-bearing mice, i.t. treatment with SZU-101 in combination with i.p. treatment with JQ-1 suppressed the growth of tumors at both injected and uninjected sites. Combination therapy increased M1/M2 ratio in TAMs, decreased PD-L1 expression and promoted the recruitment of activated CD8+ T cells in the TME. In summary, the improved therapeutic efficacy of the novel combination therapy appears to be feasible for the treatment of a diversity of cancers.

Keywords: BRD4; TAM; TLR7; antitumor; immunity.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Combination administration of SZU-101 and JQ-1 inhibited tumor growth at both injected and uninjected sites. (A) Dose optimization studies of i.t. administration of SZU-101. Balb/c mice (n = 5–7/group) were implanted with 2 × 105 4T1 cells in the right flank on Day 0, and i.t. treated with 3, 10 or 30 mg/kg SZU-101 for successive 5 days from Day 7 to Day 11. (B) Schedule optimization studies of administration of 10 mg/kg SZU-101. Balb/c mice (n = 5–7/group) were implanted with 2 × 105 4T1 cells in the right flank on Day 0 and treated with 10 mg/kg SZU-101 for different schedules (i.t. or i.p., continuous or discontinuous), as shown in the experimental protocol. (C–E) Combination therapy with SZU-101 and JQ-1. Balb/c mice (n = 7–8/group) were implanted with 2 × 105 4T1 cells in both flanks and i.t. treated with SZU-101 and i.p. treated with JQ-1, as shown in the experimental protocol. Tumor volumes (C) and tumor weights (D) at both injected and uninjected sites were monitored. Survival curves of the mice (E) were also recorded. Data represent mean ± SE, * p < 0.05, ** p < 0.01, *** p < 0.001. Tumor growth curves were analyzed by two-way ANOVA with Bonferroni post hoc test. Tumor weights were analyzed by one-way ANOVA with Tukey’s post hoc test. Survival curves were analyzed by log rank test.
Figure 2
Figure 2
Combination administration of SZU-101 and JQ-1 increased M1/M2 ratio in TAMs. (A,B) M1/M2 ratio after compound treatment. Balb/c mice (n = 5–6/group) were treated with SZU-101 and JQ-1 as described before. Tumors were harvested and tumor-infiltrating cells were analyzed by flow cytometry, and M1/M2 ratios in TAMs of both injected and uninjected sites were monitored on Day 12 (A) and 21 (B). TAMs were gated on CD45+CD11b+F4/80+ population, and M1/M2 was calculated as the percentage of M1 subset (CD206−) divided by M2 subset (CD206+) in CD45+CD11b+F4/80+ subset. (C,D) Kinetics of M1 and M2 population after compound treatment. M1 (MHC+CD206− subset) and M2 macrophages (MHC−CD206+ subset) of the injected site were monitored on Day 12 (C) and Day 21 (D). Data represent mean ± SE, * p < 0.05 (one-way ANOVA with Tukey’s post hoc test).
Figure 3
Figure 3
Combination administration of SZU-101 and JQ-1 suppressed PD-L1 expression in tumor cells. (A) Balb/c mice (n = 5–6/group) were treated with SZU-101 and JQ-1 as described before. PD-L1 levels of tumor cells (CD45− subset) of both injected and uninjected sites were monitored on Day 21. (B) Representative images of immunohistochemical staining of tumors on Day 21 for PD-L1 (Scale bars, 50 μm). Data represent mean ± SE, * p < 0.05 (one-way ANOVA with Tukey’s post hoc test).
Figure 4
Figure 4
Combination administration of SZU-101 and JQ-1 increased CD8+ T cells in spleens and TILs. (A) Balb/c mice (n = 5–6/group) were treated with SZU-101 and JQ-1 as described before. Spleens were harvested on Day 21, and T cells in spleens were analyzed by flow cytometry. Numbers of CD45+CD3+CD8+ and CD8+IFNγ+ T cells per spleen were recorded and plotted. (B) Cytotoxic T cell responses were determined on Day 21 by incubating spleen lymphocytes (effectors) with 4T1 cells (targets) at the ratio of cell number of 50:1. (C) Tumors were harvested on Day 21, and TILs of both injected and uninjected sites were analyzed by flow cytometry. Tumor-infiltrating CD8+ T cells were gated on CD45+CD3+CD8+ population, and numbers of CD8+ and CD8+IFNγ+ T cells were recorded and plotted per tumor volume (mm3). (D) Representative images of immunohistochemical staining of tumors on Day 21 for CD8 (Scale bars, 50 μm). Data represent mean ± SE, * p < 0.05, ** p < 0.01 (one-way ANOVA with Tukey’s post hoc test).
Figure 5
Figure 5
Depletion of CD8+ cells abrogated the antitumor effects of combination administration of SZU-101 and JQ-1. (A) Experimental protocol of CD8+ cell depletion. Balb/c mice (n = 5–6/group) were treated with SZU-101 and JQ-1 as described before, and anti-CD8 mAb or isotype control was injected 6 times. (B,C) Tumor volumes (B) and tumor weights (C) at both injected and uninjected sites were monitored. Data represent mean ± SE, ** p < 0.01, *** p < 0.001, ns, statistically non-significant. Tumor growth curves were analyzed by two-way ANOVA with Bonferroni post hoc test. Tumor weights were analyzed by one-way ANOVA with Tukey’s post hoc test.
Figure 6
Figure 6
Combination administration of SZU-101 and JQ-1 inhibited tumor metastasis. (A) Experimental protocol of tumor lung metastasis. Balb/c mice (n = 4–8/group) were intravenously injected through the tail vein with 5 × 104 4T1 cells on Day 0, and i.p. treated with SZU-101 and JQ-1. (B) Numbers of lung nodules were counted on Day 21. (C) Survival curves of the mice were also recorded. (D) Spleens were harvested on Day 21, and T cells in spleens were analyzed by flow cytometry. Numbers of CD45+CD3+CD8+ and CD8+IFNγ+ T cells per spleen were recorded and plotted. (E) Cytotoxic T cell responses were determined on Day 21 by incubating spleen lymphocytes (effectors) with 4T1 cells (targets) at the ratio of cell number of 50:1. Data represent mean ± SE, * p < 0.05, ** p < 0.01. Survival curves were analyzed by log rank test. Other experiments were analyzed by one-way ANOVA with Tukey’s post hoc test.
Figure 7
Figure 7
Schematic illustration of combination therapy of SZU-101 and JQ-1 on the suppression of tumor growth.
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