Tubeimoside II potentiates anti-PD-1 therapy in NSCLC by increasing ferroptosis sensitivity and remodeling immune landscape.

[BACKGROUND] Tubeimoside Ⅱ (Tub Ⅱ), a pharmacologically active triterpenoid saponin purified from tubeimu, has exhibited promising antitumor properties, yet its molecular mechanisms in NSCLC remain incompletely understood.

[METHODS] Flow cytometry and immunofluorescence were used to detect the key immune cells. Multi-omics approach, integrating proteomic profiling, single-cell RNA sequencing, and bioinformatic analyses to uncover Tub Ⅱ' s mechanisms. Target engagement was validated via DARTS, CETSA, dual luciferase reporter, Chip-PCR, and ubiquitination assays. CRISPR-Cas9 of HERC2 and JAK2 was performed to dissect their roles in Tub II-mediated effects. Preclinical models included NSCLC xenografts, lung metastasis, PDOs, and PDXs.

[RESULTS] Tub Ⅱ suppressed NSCLC cell growth by increased ROS generation, lipid peroxidation, ferrous iron content. Additionally, it enhanced antitumor T-cell immunity, promoted DC maturation and M1-like macrophage polarization, while reducing the immunosuppressive infiltration of MDSCs and Treg cells. Mechanistically, Tub Ⅱ bound to HERC2, impairing NCOA4 ubiquitination and degradation, thereby inducing ferritinophagy-dependent ferroptosis. Moreover, Tub Ⅱ targeted JAK2, inhibiting the JAK2/STAT3 pathway, leading to immune remodeling. Significantly, Tub Ⅱ synergized with chemotherapy, targeted therapy, and immunotherapy agents, enhancing antitumor efficacy in PDOs and PDXs.

[CONCLUSIONS] Our study demonstrates that Tub Ⅱ triggers ferroptosis and reprograms the immune microenvironment in NSCLC by hyperactivating HERC2-mediated ferritinophagy and suppressing the JAK2/STAT3/PDL1/CXCL12 pathway. These results not only reveal a mechanism-driven strategy to target ferroptosis and potentiate PD-1 blockade but also highlight Tub Ⅱ as a multi-targeting natural compound with promising therapeutic potential for NSCLC.