Haibang Hao

Abstract Icaritin, a hydrolytic product of icariin isolated from traditional Chinese herbal medicine genus Epimedium , has many pharmacological and biological activities. Here, we show that icaritin can effectively decrease tumor burden of murine B16F10 melanoma and MC38 colorectal tumors in a T‐cell dependent manner. The treatment effects are associated with increased CD8 T‐cell infiltration and increased effector memory T‐cell frequency. In vivo depletion of CD8 T cell using an anti‐CD8 monoclonal antibody abolished the antitumor effect, which supports the critical role of CD8 T cells during icaritin treatment. By analyzing immune cells in the tumor tissue, we found reduced frequency of CD11b + Gr1 + myeloid‐derived suppression cells (MDSCs) infiltration and downregulation of PD‐L1 expression on MDSCs after icaritin treatment. This was not limited to MDSCs, as icaritin also decreased the expression of PD‐L1 on neutrophils. Importantly, the combination of anti‐PD‐1/CTLA‐4 and icaritin significantly enhances antitumor ability and increases the efficacy of either treatment alone. Our findings reveal that icaritin induces antitumor immunity in a CD8 T‐cell‐dependent way and justify further investigation of combining immune checkpoint therapy to icaritin‐based antitumor therapy.

Icaritin, a small molecule currently being investigated in phase III clinical trials in China (NCT03236636 and NCT03236649) for treatment of advanced hepatocellular carcinoma (HCC), is a prenylflavonoid derivative obtained from the Epimedium genus. Previously, it was found that Icaritin decreased the expression of PD-L1, but its direct molecular targets and the underlying mechanisms have not been identified. In this study, we report the identification of IKK-α as the protein target of Icaritin by biotin-based affinity binding assay. The further mutagenesis assay has provided evidence that C46 and C178 in IKK-α were essential amino acids for Icaritin binding to IKK-α, revealing the binding sites of Icaritin to IKK-α for the first time. Functionally, Icaritin inhibited the NF-κB signalling pathway by blocking IKK complex formation, which led to decreased nuclear translocation of NF-κB p65, and subsequent downregulation of PD-L1 expression in a dose-dependent manner. More importantly, PD-L1-positive patients exhibited longer overall survival upon Icaritin therapy. Finally, Icaritin in combination with checkpoints antibodies, such as α-PD-1, has demonstrated much better efficacy than any single therapy in animal models. This is the first report that anticancer effects of Icaritin are mediated, at least in part, by impairing functions of IKK-α.

Akkermansia (Akk) is a commensal bacterium in gut microbiota known to enhance anti-tumor immunity and improve responses to immunotherapy. However, the clinical translation of Akk-based therapies still faces critical challenges, including intestinal colonization difficulties, inter-patient variability, and safety risks. Here, we identified the flavonoid icariin as a novel prebiotic candidate that could specifically enrich intestinal Akk abundance under various conditions both in vivo and in vitro. Oral administration of icariin selectively increased intestinal Akk abundance in tumor-bearing and non-tumor mice, regardless of their immunocompetent or immunodeficient status. Moreover, in vitro assays confirmed that icariin promoted Akk growth in a mucin-dependent manner. Transcriptomic and metabolomic analyses revealed that icariin enhanced mucin-associated metabolic pathways to support Akk growth. Mechanistically, we found that icariin increased the enzymatic activity of N-acetylgalactosaminidase Amuc_0920 by stabilizing key residues at the binding sites for the substrate GalNAc, which enhanced mucin catabolism and promoted Akk growth. Functionally, we further found that this Akk enrichment enhanced intratumoral CD8+ T cell functions by single-cell RNA sequencing. Consequently, icariin-induced Akk enrichment significantly enhanced the efficacy of anti-PD-1 immunotherapy in tumor mouse models. These findings establish icariin as a promising Akk-targeting prebiotic that selectively enriches Akk to improve cancer immunotherapy.