Background
Radiation therapy (RT), the standard-of-care for glioblastoma, activates cancer cell-intrinsic type I interferon (IFN-I) responses and generates systemic immunity in multiple cancers. However, the combination of RT and immune checkpoint blockers (ICB) failed to improve glioblastoma patient survival, suggesting the presence of potent immunosuppressive mechanisms. We recently demonstrated that RT induces a metabolic shift towards fatty acid synthesis to maintain glioblastoma survival; partly mediated by the fatty acid synthase (FASN). FASN-mediated lipid synthesis has been shown to inhibit IFN-I responses in the context of viral immunity. Therefore, we hypothesize that blocking FASN can enhance IFN-I and restore ICB sensitivity in irradiated glioblastoma.
Methods
In vitro, FASN was targeted using shRNA, CRISPR/Cas9, and FASN inhibitors (FASNi) in murine glioblastoma cells (GL261 and CT2A), and IFN-I secretion was quantified by ELISA. Key nucleic acid sensors (cGAS, RIG-I, and MDA5) were blocked to determine the IFN-I modulation mechanism in FASN-blocked irradiated cells. In vivo, CT2AshNS and CT2AshFASN cells were intracranially injected and received selective irradiation (10Gy) on day 10. Immune infiltration was analyzed using spatial multiplexing and immunofluorescence (n=3/group) on day 17. Additionally, the ability of FASN blockade to sensitize irradiated glioblastoma to anti-PD-1 was assessed in glioblastoma-bearing mice treated with FASNi (daily, i.p. days 9–30), anti-PD-1 (bi-weekly, i.p., days 15–24), and clinically relevant radiation (5 fractions of 6Gy). Mice were followed up for survival (n=7/group).
Results
Our findings demonstrate that targeting FASN using orthogonal methods significantly enhanced the secretion of IFN-beta and CXCL10 in irradiated glioblastoma cells. Interestingly, inhibition of cGAS, RIG-1 or MDA5 prevented IFN-I in irradiated FASN-blocked glioblastoma cells. In vivo experiments revealed a significant increase in the infiltration of CD11c+ cells and CD8+ T cells within irradiated CT2AshFASN tumors compared to CT2AshNS; an effect that was lost when IFN-I pathway was blocked with an anti-IFNAR antibody. Most importantly, FASNi prolonged the survival of mice undergoing combined treatment of RT+anti-PD-1, with 86% of mice surviving up to 60 days post tumor implantation as opposed to 43% in mice treated with RT, RT+FASNi or RT+anti-PD-1.
Conclusions
Our study highlights the role of FASN in shaping the immunogenicity of irradiated glioblastoma. By targeting FASN, we restored the ability of nucleic acid sensors to induce IFN-I in irradiated glioblastoma, therefore promoting immune cell infiltration and sensitizing irradiated glioblastoma to anti-PD-1. These findings shed the light on FASN as an attractive target to unleash the immuno-stimulatory properties of RT in glioblastoma.
Ethics Approval
Mice experiments were approved by the Institutional Animal Care and Use Committee #2019–0042.