Cucurbitacin B stimulates PD-1 immunotherapy response in malignant breast cancer by covalent targeting MTCH2

Abstract

Background: Effective therapies for malignant breast cancer are urgently needed, as resistance and immunosuppressive microenvironments limit PD-1 blockade efficacy. The natural product Cucurbitacin B (CuB) reportedly sensitizes breast cancer to PD-1 immunotherapy, yet its molecular mechanism is undefined.

Purpose: Here, we sought to identify the direct molecular targets of CuB and elucidate the mechanisms responsible for its synergy with PD-1 blockade in breast cancer.

Study design and methods: We used Quantitative Thiol Reactivity Profiling (QTRP) to identify CuB-binding proteins. Binding interactions were validated using microscale thermophoresis (MST), cellular thermal shift assay (CETSA), and activity-based protein profiling (ABPP). The functional outcomes of CuB-protein interactions were explored using in vitro, ex vivo, and in vivo models, including cell lines, tumor organoids, and animal models of invasive breast cancer.

Results: We identified the mitochondrial outer membrane protein MTCH2, often overexpressed in aggressive breast cancer, as a direct covalent target of CuB. CuB binding to MTCH2 disrupted mitochondrial integrity, causing mitochondrial DNA (mtDNA) release into the cytosol and subsequent activation of the cGAS-STING innate immune pathway. This culminated in type I interferon production, activation of tumor-associated neutrophils, and enhanced anti-tumor immunity. Co-administration of CuB and PD-1 blockade demonstrated significant synergistic efficacy in preclinical breast cancer models.

Conclusions: This work elucidates a novel mechanism by which CuB enhances anti-tumor immunity: covalent targeting of MTCH2 triggers mitochondrial dysfunction and cGAS-STING pathway activation. Our findings establish MTCH2 as a key node linking mitochondrial function to tumor immunogenicity and provide a rationale for combining CuB, or potentially MTCH2 modulators, with PD-1 blockade for treating malignant breast cancer.

Keywords: Aggressive breast cancer; Cucurbitacin B; MTCH2; Tumor-associated neutrophils; mtDNA-cGAS-STING; Ιmmunotherapy response.