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Purpose: Sphingosine-1-phosphate (S1P) is a pleotropic bioactive lipid mediator involved in the pathogenesis of inflammation and cancer. FTY720 is a “functional antagonist” of S1P by binding and induces the internalization and degradation of S1P receptors. Indeed, FTY720 was shown to have antitumor activity in several tumor models, while whether its immune-suppressive activity promotes or inhibits tumor growth is still unclear. In this study, we explored the antitumor activity of FTY720 in various cancer cells and its underlying mechanisms. Experimental Procedures: We analyzed the antiproliferative activity of FTY720 in nearly 30 human cancer cell lines and 6 mouse cancer cell lines. Human colon cancer cells SW620 and mouse breast cancer cells 4T1, which are sensitive to FTY720, were selected and used for in vivo study. SW620 was subcutaneously inoculated into Balb/c nude mice and 4T1 were in situ inoculated into immune-competent Balb/c mice. Mice was administrated intraperitoneally with low dose (0.5 mg/kg/d) and high dose (5 mg/kg/d) of FTY720. Tumor volume and body weight were measured every 3 days. After 3 weeks, the mice were sacrificed, and the tumor was removed and weighed, and kept for further analysis. The signaling pathway involved in S1P pathway and neovascularization were analyzed. In addition, the pharmacokinetics of FTY720 was also determined in mice. The effect of FTY720 on angiogenesis was also analyzed at cellular level, as well as the gene expression in HUVEC cells. Summary: FTY720 showed potent antiproliferative activity across various cancer cell lines. The IC50 for SW620 and 4T1 was 3.71 μM and 3.38 μM respectively, which were most sensitive. In animal study, both low-dose and high-dose FTY720 dramatically inhibited the tumor growth in SW620 xenograft model, while only high-dose FTY720 inhibited tumor growth in 4T1 xenograft model. Since the serum concentration of FTY720 was much lower than its cytotoxicity at low dose, we thus propose the anti-umor activity of FTY720 may due to its antiangiogenic activity. Moreover, the antitumor activity of FTY720 was much lower for 4T1 as compared to SW620 xenograft model, which possibly is caused by its immune-suppressive activity as it depleted the circulating lymphocytes. Besides, FTY720 dramatically inhibited neovascularization in tumor tissues. The half-life and AUClast are good for FTY720 in mice. FTY720 inhibited new vessel formation at cellular level. In addition, the microarray data showed the low-dose FTY720 (100 nM) induced the alteration of a few genes’ expression. Conclusion: FTY720 has potent antitumor activity both in vitro and in vivo, the mechanism of which is associated with proapoptotic, antiangiogenic and anti-inflammatory activities. The immune-suppressive activity of FTY720 may limit the antitumor activity in immune-competent individuals. This study provides a comprehensive understanding of the immune-suppressant FTY720 in antitumor. Note: This abstract was not presented at the conference. Citation Format: Zuoquan Xie, Xiaoxu Chen, Hui Xu, Jing Ai, Meiyu Geng. In vitro and In vivo antitumor activity of immune suppressant FTY720 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A83.

Purpose: Sphingosine-1-phosphate (S1P) is a bioactive lipid that promotes cell survival and proliferation, and modulation of lymphocyte egress. It plays important roles in the pathogenesis of inflammation and cancer. FTY720 is a “functional antagonist” of S1P by binding and induces the internalization and degradation of S1P receptors. FTY720 showed antitumor activity in several tumor models, but the mechanism has not been fully illustrated. In this study, we used microarray to explore the signaling pathways and genes regulated by FTY720, which may help to illustrate the mechanism of action. Experimental Procedures: We tested the inhibitory activity of FTY720 in various human cancer cell lines. Human colon cancer cells SW620, which are sensitive to FTY720, were selected and used for further study. SW620 was subcutaneously inoculated into Balb/c nude mice, then treated intraperitoneally with low dose (0.5 mg/kg/d) and high dose (5 mg/kg/d) of FTY720. Meanwhile, microarray analysis was used to explore the modulated genes by FTY720. Moreover, we tested the anti-neovascularization effect of FTY720 on endothelial cells (HMEC), which was shown to have this activity in vivo, and we also profiled the differentially expressed genes regulated by FTY720 on HMEC cells. Summary: FTY720 showed potent inhibitory activity on various cancer cell lines. The colon cancer cell SW620 was one of the most sensitive cells in response to FTY720 with an IC50 of 3.71 μM. FTY720 also inhibited new vessel formation at cellular level. In animal study, both low-dose and high-dose FTY720 dramatically inhibited the tumor growth in SW620 xenograft model. Since the serum concentration of FTY720 was much lower than its cellular cytotoxicity at low dose, we propose the antitumor activity may not only be due to a direct effect on tumor growth, but also on neovascularization and lowering of circulating lymphocytes. In transcriptomic study, we found cell cycle and DNA repair pathways were downregulated by FTY720 both in SW620 and HMEC cells. In HMEC cells, mTORC1, MYC, P53, FOXO1, and immune response pathways were downregulated while steroid metabolism and terpenoid backbone biosynthesis pathways were upregulated by FTY720. In SW620 cells, fatty acid metabolism, FOXO1, inflammatory and immune response pathways were downregulated while steroid metabolism and terpenoid backbone biosynthesis pathways were upregulated by FTY720. Thus, there are many overlaps of regulated pathways and genes by FTY720 between SW620 and HMEC cells. Interestingly, the angiogenesis and blood vessel morphogenesis were both downregulated by FTY772 in SW620 and HMEC cells. Conclusion: FTY720 has potent antitumor activity in vitro and in vivo. It showed pleiotropic effects on gene expression, especially downregulating those genes involved in cell cycle, angiogenesis, and inflammatory and immune response. This study displayed a landscape of differentially expressed genes modulated by FTY720 and provided a basis for further mechanistic study. Citation Format: Zuoquan Xie, Xiaoxu Chen, Qingzhong Zeng, Meiyu Geng. Transcriptomic landscape of the immune suppressant FTY720 in antitumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B61.

The interaction between tumor cells and their immunosuppressive microenvironment promotes tumor progression and drug resistance. Thus, simultaneously targeting tumor cells and stromal cells is expected to have synergistic antitumor effects. Herein, we present the preclinical antitumor investigation of 3D185, a novel dual inhibitor targeting FGFRs, which are oncogenic drivers, and CSF-1R, which is the major survival factor for protumor macrophages. The antitumor characteristics of 3D185 were assessed by a range of assays, including kinase profiling, cell viability, cell migration, immunoblotting, CD8+ T-cell suppression, and in vivo antitumor efficacy, followed by flow cytometric and immunohistochemical analyses of tumor-infiltrating immune cells and endothelial cells in nude mice and immune-competent mice. We found that 3D185 significantly inhibited the kinase activity of FGFR1/2/3 and CSF-1R, with equal potency and high selectivity over other kinases. 3D185 suppressed FGFR signaling and tumor cell growth in FGFR-driven models both in vitro and in vivo. In addition, 3D185 could inhibit the survival and M2-like polarization of protumor macrophages, reversing the immunosuppressive effect of macrophages on CD8+ T cells as well as CSF1-differentiated macrophage induced-FGFR3-aberrant cancer cell migration. Furthermore, 3D185 inhibited tumor growth via remodeling the tumor microenvironment in TAM-dominated tumor models. In summary, 3D185 is a dual inhibitor against FGFR1/2/3 and CSF-1R that exhibits potent antitumor activities. This compound is promising because it simultaneously targets tumor cells per se and the immunosuppressive tumor microenvironment to synergistically antagonize tumors. Our study provides a solid foundation for the investigation of 3D185 in cancer patients, particularly in patients with aberrant FGFR and abundant macrophages, who respond poorly to classic pan-FGFRi treatment. Now 3D185 is in phase I clinical trials. Citation Format: Yang Dai, Yichun Ji, Yanyan Shen, Yi Su, Bo Liu, Yueliang Wang, Deqiao Sun, Yuchen Jiang, Chuantao Zha, Zuoquan Xie, Jian Ding, Meiyu Geng, Jing Ai, Xia Peng, Pengcong Hou, Yi Chen. Preclinical evaluation of 3D185, a novel potent inhibitor of FGFR1/2/3 and CSF-1R, in FGFR-dependent and macrophage-dominant cancer models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A78.