Isovalerylspiramycin I Reprograms the Immunosuppressive and Temozolomide-Resistant Microenvironment by Inhibiting the Frizzled-5/Wnt/β-Catenin Pathway in Glioblastoma.

Abstract

Glioblastoma (GBM), the most prevalent and lethal primary brain malignancy in adults, currently lacks treatment effective options. Repurposing existing pharmaceutical agents as novel therapeutic modalities represents a viable strategy for efficiently utilizing resources. Here, we demonstrated that Isovalerylspiramycin I (ISP-I), the active component of a novel macrolide antibiotic, exerts a synergistic effect with temozolomide (TMZ) to enhance anti-GBM efficacy. ISP-I potently induced cytotoxicity and apoptosis through the induction of DNA double-strand breaks. The synergistic activity (combination index < 1) was confirmed for ISP-I in combination with TMZ against GBM. Additionally, ISP-I was found to induce immunogenic cell death, as evidenced by increased adenosine triphosphate release and calreticulin exposure. In murine models, ISP-I increased tumor-infiltrating CD8⁺ T cells, enhanced effector subsets, and reduced exhausted subsets. Mechanistically, ISP-I targeted the Frizzled-5 (FZD5)/Wnt/β-catenin signaling pathway, resulting in suppression of GSK-3β phosphorylation. This event subsequently increased β-catenin phosphorylation, reducing its translocation into the nucleus. Consequently, the binding of transcription factors (T-cell factor 1/lymphoid enhancer factor 1) to promoters of CD274 and O⁶-methylguanine-DNA methyltransferase (MGMT) was impeded, thereby enhancing GBM cell susceptibility to TMZ. These findings elucidate the mechanisms underlying ISP-I's therapeutic efficacy in GBM and provide essential evidence for its clinical translation and combinatorial therapeutic strategies.