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

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.

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.

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.

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.

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.