SKI-G-801, an AXL kinase inhibitor, blocks metastasis through inducing anti-tumor immune responses and potentiates anti-PD-1 therapy in mouse cancer models

Abstract

Objectives: AXL-mediated activation of aberrant tyrosine kinase drives various oncogenic processes and facilitates an immunosuppressive microenvironment. We evaluated the anti-tumor and anti-metastatic activities of SKI-G-801, a small-molecule inhibitor of AXL, alone and in combination with anti-PD-1 therapy.

Methods: In vitro pAXL inhibition by SKI-G-801 was performed in both human and mouse cancer cell lines. Immunocompetent mouse models of tumor were established to measure anti-metastatic potential of SKI-G-801. Furthermore, SKI-G-801, anti-PD-1 or their combination was administered as an adjuvant or neoadjuvant in the 4T1 tumor model to assess their potential for clinical application.

Results: SKI-G-801 robustly inhibited pAXL expression in various cell lines. SKI-G-801 alone or in combination with anti-PD-1 potently inhibited metastasis in B16F10 melanoma, CT26 colon and 4T1 breast models. SKI-G-801 inhibited the growth of B16F10 and 4T1 tumor-bearing mice but not immune-deficient mice. An antibody depletion assay revealed that CD8⁺ T cells significantly contributed to SKI-G-801-mediated survival. Anti-PD-1 and combination group were observed the increased CD8⁺Ki67⁺ and effector T cells and M1 macrophage and decreased M2 macrophage, and granulocytic myeloid-derived suppressor cell (G-MDSC) compared to the control group. The neoadjuvant combination of SKI-G-801 and anti-PD-1 therapy achieved superior survival benefits by inducing more profound T-cell responses in the 4T1 syngeneic mouse model.

Conclusion: SKI-G-801 significantly suppressed tumor metastasis and growth by enhancing anti-tumor immune responses. Our results suggest that SKI-G-801 has the potential to overcome anti-PD-1 therapy resistance and allow more patients to benefit from anti-PD-1 therapy.

Keywords: AXL; immunotherapy; kinase inhibitor; metastasis; small molecule.

Figure 1

SKI‐G‐801 is a novel AXL kinase inhibitor. (a) Crystal structure of SKI‐G‐801 in AXL kinase domain (left). Chemical structure of SKI‐G‐801 (right). (b) Western blot analysis of pAXL inhibition by SKI‐G‐801 and R428 in MDA‐MB‐231 and Hs578T cells. The experiment was repeated twice. (c) MDA‐MB‐231 cells were incubated with either SKI‐G‐801 or R428 at the indicated concentrations for 48 h, followed by an invasion assay. The experiment was repeated twice. (d) B16F10 cells (1 × 10⁶ cells per mouse, n = 3 per group, derived from two independent experiments) were injected intravenously. SKI‐G‐801 (30 mg kg⁻¹ day⁻¹) was administered from day −1 to day +14. The mice were sacrificed to evaluate the metastasis. (e) CT26‐luciferase cells (1 × 10⁴ cells per mouse, n = 5 per group, derived from two independent experiments) were implanted into the intraperitoneal cavity. SKI‐G‐801 (30 mg kg⁻¹, once a day) was administered from day −1 to day +15. Luciferase expression was determined on days 0, 3, 6, 9, 12 and 15 using the Xenogen in vivo imaging system.

Figure 2

Combination of SKI‐G‐801 and anti‐PD‐1 synergistically inhibits spontaneous lung metastasis in the 4T1 model. (a) Western blot analysis for pAXL inhibition by SKI‐G‐801 in 4T1 cells. Experiments were repeated twice. (b) pAXL were measured in 4T1 tumor‐bearing syngeneic mice. Mice were treated with SKI‐G‐801 10 mg kg⁻¹ (upper) and 30 mg kg⁻¹ (lower) for up to 24 h. (c) Representative images of H&E‐stained lung tissue demonstrating lung metastases are shown. (d) Total number of metastatic nodules was counted, and tumor area in lung tissue was measured. Lung and percentage of tumor area were calculated with ImageJ (n = 4 or 6 per group, derived from two independent experiments). (e) The proportions of IFN‐γ‐expressing cytotoxic T cells (CD3⁺CD8⁺IFN‐γ⁺) and central memory CD8⁺ T cells (CD3⁺CD8⁺CCR7⁺), measured by flow cytometry, were significantly increased in the combination group compared to other treatment groups. The dendritic cells (DCs) population (F4/80⁻CD11c⁺H2⁺) and CD80 expression on DCs are shown. Values are indicated as means ± SEM, and P‐values were calculated using the Student’s t‐test; *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. Flow cytometry analysis was done once.

Figure 3

CD8⁺ T cells mediate the therapeutic effects of SKI‐G‐801. (a) B16F10 and 4T1 cells were separately implanted in immunocompetent or immunodeficient (nude) mice, and subsequently, the mice were treated with 30 mg kg⁻¹ SKI‐G‐801 for the indicated number of days (n = 5 per group, derived from one independent experiment). Vehicle‐treated mice are marked in black (■), and SKI‐G‐801‐treated mice are marked red (). Significance was determined using the log‐rank test; *P ≤ 0.05. (b) Overall survival rate was monitored in SKI‐G‐801‐treated 4T1 tumor‐bearing mice following injection of anti‐CD8, anti‐NK and anti‐CD4 antibodies, and IgG control (n = 5 per group, derived from one independent experiment). Survival was determined by either death or high tumor volume (= 2500 mm³). Significance was determined by the log‐rank test; **P ≤ 0.01.

Figure 4

SKI‐G‐801 inhibits CT26 tumor growth by promoting anti‐tumor immunity. (a) Tumor growth was measured after mono‐ or combination therapy [anti‐PD‐1 (10 mg kg⁻¹, intraperitoneal injection, biweekly), SKI‐G‐801 (30 mg kg⁻¹, orally, once a day), SKI‐G‐801 + anti‐PD‐1] (n = 9 per group, derived from one independent experiment). Values are indicated as means ± SEM, and P‐values were calculated using the Student’s t‐test; *P ≤ 0.05 and ***P ≤ 0.001. (b) Tumor growth inhibition (TGI) was measured at day 17; in combination group, 4 of 9 mice showed > 90% of TGI. (c) Survival rate was monitored in each group till the end of experiment. Survival was determined by either death or high tumor volume (= 2500 mm³). Significance was determined by the log‐rank test; *P ≤ 0.05 and **P ≤ 0.01. (d) Immune cell population was analysed by flow cytometry. The gating strategy is shown (Supplementary figure 7). Significantly increased number of proliferating CD8⁺ T_c cells, effector T_c cells and M1 macrophages were observed in combination group compared to vehicle group. Effector memory T_c cells in SKI‐G‐801 group showed significant increase compared to vehicle group. G‐MDSCs in both SKI‐G‐801 single and combination group decreased significantly compared to the vehicle group. Values are indicated as means ± SEM, and P‐values were calculated using the Student’s t‐test; *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. Flow cytometry analysis was done once.

Figure 5

Neoadjuvant combination of anti‐PD‐1 and SKI‐G‐801 treatment most significantly increased survival of mice‐harbouring 4T1 tumors. (a, b) Scheme of neoadjuvant and adjuvant treatment, respectively. Neoadjuvant and adjuvant experiments were performed together, but survivals are displayed separately for readability (n = 4 or 5 per group, derived from one independent experiment). Mice were inoculated with 5 × 10⁴ 4T1 cells subcutaneously on day 0, and tumors were removed surgically on day 17. Drugs were administered either before (neoadjuvant) or after surgery (adjuvant) for 6 days. Survival was determined by death. Mice that died during or within 1 week of surgery (considered as death from surgery) were excluded. There were no deaths because of developed primary tumor regrowth (considered as death from tumor burden). Statistical significance was determined by the log‐rank test; *P ≤ 0.05, **P ≤ 0.01. (c) Three tumors per group were stained, and CD3⁺ and PD‐L1⁺ cells per unit area (mm⁻¹) or percentage (%) were analysed using Vectra Polaris (left). Nuclei are marked in blue (), CD3⁺ cells are marked in pink (), and PD‐L1⁺ cells are marked in yellow (). IHC was done once and CD3⁺ cells (per mm²), and percentage of PD‐L1⁺ cells (%) were calculated (right). Values are indicated as means ± SEM, and P‐values were calculated using the Student’s t‐test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.