Protein Kinase Inhibitor-Mediated Immunoprophylactic and Immunotherapeutic Control of Colon Cancer

Abstract

Immunotherapy has allowed major advances in oncology in the past years, in particular with the development of immune checkpoint inhibitors, but the clinical benefits are still limited, particularly in colorectal cancer (CRC). Our scientific approach is based on the search for innovative immunotherapy with a final goal that aims to induce an effective antitumor immune response in CRC. Here, we focused on a multikinase inhibitor, H89. We carried out in vivo experiments based on syngeneic mouse models of colon cancer in BALB/c mice and chemically colon tumorigenesis. Flow cytometry, RNAseq, RT-qPCR, antibody-specific immune cell depletion, and Western blot were used to identify the immune cell type involved in the preventive and antitumor activity of H89. We demonstrated that H89 delays colon oncogenesis and prevents tumor growth. This latter effect seems to involve NK cells. H89 also inhibits colon tumor growth in a T-cell-dependent manner. Analysis of the immune landscape in the tumor microenvironment showed an increase of CD4⁺ Th1 cells and CD8⁺ cytotoxic T cells but a decrease of CD4⁺ T_reg cell infiltration. Mechanistically, we showed that H89 could promote naïve CD4⁺ T-cell differentiation into Th1, a decrease in T_reg differentiation, and an increase in CD8⁺ T-cell activation and cytotoxicity ex vivo. Furthermore, H89 induced overexpression of genes involved in antitumor immune response, such as IL-15RA, which depletion counteracts the antitumor effect of H89. We also found that H89 regulated Akt/PP2A pathway axis, involved in TCR and IL-15 signaling transduction. Our findings identify the H89 as a potential strategy for immune system activation leading to the prevention and treatment of CRC.

Keywords: H89; colorectal cancer; immunoprophylaxis; immunotherapy; kinase inhibitor.

Figure 1

H89 delays tumor growth and AOM/DSS-driven carcinogenesis. (A) Macroscopic evaluation of the number of colorectal polyps in BALB/c mice with AOM/DSS-induced colorectal carcinoma, treated or not with H89 (5 mg/kg, oral administration) twice a week (n = 10 mice/group). (B) Macroscopic images of mouse colons treated or not with H89. (C) To assess the preventive effect of H89 in CT26-bearing BALB/c mice, H89 (or NaCl in the control group) was injected for 3 days in a row (10 mg/kg, i.p.). On day 4, 5 × 10⁵ CT26 cells were injected in s.c., and tumor growth was monitored three times a week (n = 5). (D) Two days before the first H89 injection, mice were treated with an anti-asialoGM1 to deplete NK cells (10 µl in 100 µl of NaCl according to the supplier’s instructions). Anti-Asialo GM1 Injections were repeated during the first and the last H89 injections, and then every 5 days (n = 7 mice/group). Statistically significant differences were determined by using a t-test (A) or two-way ANOVA (C, D); ^*p ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001.

Figure 2

H89 mediates immunotherapeutic activity against colon cancer. (A, B) CT26 or C51 tumor-bearing BALB/c mice (5 × 10⁵ murine colon cancer cells in s.c.) were treated, or not (NaCl, i.p.), with H89 (10 mg/kg, i.p.) twice a week, and tumor growth was monitored three times a week (A, n = 10 mice/group; B, n = 7 mice/group). (C) Oral administration of H89 (5 mg/kg), or NaCl in the control group, twice a week in CT26 tumor-bearing BALB/c mice. Tumor growth was monitored three times a week (n = 7 mice/group). (D) 4T1 breast cancer cells tumor-bearing mice (5 × 10⁵ 4T1 cells in s.c.) were treated, or not (NaCl, i.p.), with H89 (5 mg/kg, i.p.) twice a week, and tumor growth was monitored three times a week (n = 5 mice/group). (E) CT26 tumor-bearing BALB/c mice (5 × 10⁵ CT26 cells in s.c.) were treated, or not (NaCl, i.p.), with H89 (10 mg/kg, i.p.) twice a week, in combination with 5-fluorouracil (5-FU, 5 mg/kg in i.p.) once a week, and tumor growth was monitored three times a week (n = 14 mice/group). (F) Swiss nude immunodeficient mice, bearing CT26 tumors (5 × 10⁵ CT26 cells in s.c.) were treated, or not (NaCl, i.p.), with H89 (10 mg/kg, i.p.) twice a week, and tumor growth was monitored three times a week (n = 5 mice/group). (G) CT26 tumor-bearing BALB/c mice (5 × 10⁵ CT26 cells in s.c.) were treated, or not (NaCl, i.p.), with H89 (10 mg/kg, i.p.) twice a week. Mice also received anti-CD8a or control IgG injections once a week (500 µg in i.p.), and tumor growth was monitored three times a week (n = 7 mice/group). Statistically significant differences were determined by using two-way ANOVA: ^*p ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001; ^****p ≤ 0.0001; n.s., nonsignificant results.

Figure 3

H89 increases CD8⁺ TL tumor-infiltration, activation, and function. (A, B) Flow cytometry analysis (n = 10 mice/group) of the intratumor infiltration of CD8⁺ T cells under H89 treatment on day 10 (D10), D14, and D21 after CT26 colon cancer cells injection (5 × 10⁵ in s.c.). BALB/c mice were treated or not (NaCl) by H89 (10 mg/kg, i.p. administration twice a week). (C) IHC analysis on CT26 tumors after CD8⁺ T-cell labelling at D14 and D21 (representative images of 3 animals/group/time point). RT-qPCR analysis of CXCL10 (D) and IFN-γ (E) expression at D14 and D21 after CT26 cancer cell injection (n = 3 mice) and H89 treatment (10 mg/kg, i.p., NaCl in the control group). The results are presented as the mean of 2^−ΔCT values. (F, G) Flow cytometry analysis after intracellular labeling (n = 10 mice/group) of IFN-γ and granzyme B in intratumor CD8⁺ T cells at D10, D14, and D21 after CT26 colon cancer cell injection and H89 treatment (10 mg/kg, i.p., NaCl in the control group). (H) Flow cytometry analysis of granzyme B production of splenic CD8⁺ T cells ex vivo after H89 treatment (5 µM, 72 h) (n = 3) and (I) cytotoxic assay using CD8⁺ T cells from OT-I mice cocultured with B16-OVA cells in vitro treated by H89 (5 µM, 48 h) (n = 3). (H–I) The results are calculated as the mean of 2^−ΔCT values. Each control values (NT) are set at 1 arbitrary unit (A.U.), and H89-treated conditions are compared with the control. Statistically significant differences were determined by using a t-test: ^*p ≤ 0.05; ^**p ≤ 0.01.

Figure 4

Effect of H89 on CD4⁺ TL infiltration and differentiation. (A, B, D) Flow cytometry analysis (n =10 mice/group) of the intratumor infiltration of CD4⁺ Th1, Treg, and IFN-γ expression in CD4⁺ T cells on day 10 (D10), D14, and D21 after CT26 colon cancer cell injection into BALB/c mice (5 × 10⁵ in s.c.), treated or not by H89 (10 mg/kg, i.p. injection two times a week). Control group received NaCl injection. (C) RT-qPCR analyses of Tbet expression at D14 and D21 after CT26 cancer cell injection into BALB/c mice (n =3 mice). The results are presented as the mean of 2^−ΔCT values. (E, F) RT-qPCR analyses of Tbet and FoxP3 expression on splenic naive CD4⁺ T cells from BALB mice after in vitro differentiation into Th1 and Treg under H89 treatment (1 µM, 72 h) (n = 3). The results are calculated as the mean of 2^−ΔCT values. Each control values (NT) are set at 1 arbitrary unit (A.U.) and H89-treated conditions are compared with control. Statistically significant differences were determined by using a t-test: ^*p ≤ 0.05.

Figure 5

H89 modulates immunosuppressive receptors expressed either by T cells or colon cancer cells. Flow cytometry analysis of PD-1 expression on CD4⁺ T cells isolated from the spleen of BALB/c mice (A) or MOLT-4 cells (B) cells treated by H89 for 24 h (n = 3). Flow cytometry intracellular K⁺ detection using APG-4 probe on splenic CD4⁺ T cells treated by H89 for 2 h (n = 3) (C). Flow cytometry analysis of PD-L1/CD80 expression on CT26 colon cancer cells treated by H89 for 24 h (n = 3) (D, E). Statistically significant differences were determined by using a t-test: ^*p ≤ 0.05; ^**p ≤ 0.01.

Figure 6

H89 regulates signaling pathways involved in immune cell activation and cancer cell growth. (A) RNAseq analysis were performed on CT26 solid tumors isolated from BALB/c mice treated or not (NaCl) with H89 (10 mg/kg, i.p.) at D14 after colon cancer cells injection (n = 3 mice/group). (B) RT-qPCR analysis of IL-15 expression mRNA level at D14 and D21 after CT26 cancer cell injection into BALB/c mice (n = 3 mice/group) and ELISA analysis of IL-15 on tumor lysates ad D14 (n = 5 mice/group). (C) RT-qPCR analysis of IL-15 expression mRNA level on CT26 cells treated in vitro with H89 for 24 h (n = 3). (D) CT26 tumor-bearing BALB/c mice (5 × 10⁵ CT26 cells in s.c.) were treated, or not (NaCl, i.p.), with H89 (10 mg/kg, i.p.) twice a week. Mice also received anti-TMB1 or control IgG injections once a week (50 µg in i.p.), and tumor growth was monitored three times a week (n = 7 mice/group). (E–G) Western blot analysis of Akt phosphorylation (P-Akt) after H89 treatment in CD8 T cells isolated from the spleen of BALB/c mice or MOLT-4 and Jurkat T cells (n = 5) (H, I) Quantification of the enzymatic activity of phosphatase PP2A in Jurkat and MOLT-4 T cells (n = 3). Statistically significant differences were determined by using two-way ANOVA (D) or a t-test (B, C, E–I): ^*p ≤ 0.05 (or 0.1 in (B) with 90% confidence level); ^**p ≤ 0.01; ^***p ≤ 0.001; n.s., nonsignificant results. RNAseq analysis was performed with the DESeq2 package using a Wald test.