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

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 <i>CD274</i> and O⁶-methylguanine-DNA methyltransferase (<i>MGMT</i>) 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.