Pulsatilla saponin D triggers SHP2-mediated mitochondrial dysfunction and ferroptosis to augment cancer immunotherapy

Despite revolutionizing cancer treatment, immune checkpoint therapy (ICT) shows limited efficacy, primarily due to the non-immunogenic and immunosuppressive microenvironments. Identifying small-molecule inducers of immunogenic cell death (ICD) from natural sources represents a compelling strategy to overcome this therapeutic barrier.

This study aimed to identify natural ICD inducers and characterize their mechanisms in potentiating ICT efficacy.

High-throughput screening of a natural product library was performed to identify candidate ICD inducers. Pulsatilla saponin D (PSD), a triterpenoid saponin isolated from Pulsatilla koreana, was selected for comprehensive mechanistic and therapeutic evaluation. ICD induction was evaluated by measuring the release of damage-associated molecular patterns (DAMPs). The direct intracellular target of PSD was identified by limited proteolysis-mass spectrometry (LiP-SMap). Downstream mechanistic alterations in mitochondrial dynamics, mitochondrial reactive oxygen species (mtROS) generation, and hallmarks of ferroptosis were comprehensively characterized.

PSD robustly elicited the release of DAMPs, signifying enhanced immunogenic stress. In both in vitro and in vivo models, PSD treatment significantly inhibited tumor progression by promoting the maturation of dendritic cells (DCs) and enhancing antigen cross-presentation, which subsequently invigorated CD8⁺ T-cell responses. Mechanistically, PSD binds directly to SHP2 (Src homology region 2 domain-containing phosphatase 2), impairing DRP1 oligomerization and inducing mitochondrial dysfunction. This cascade leads to an accumulation of mtROS and accelerated lipid peroxidation, ultimately executing ferroptotic cell death. Notably, the combination of PSD with anti-PD-1 blockade demonstrated a synergistic antitumor effect, characterized by an increased infiltration of cDC1 and cytotoxic T cells, alongside a reduction in immunosuppressive populations.

Our work implicates SHP2-mediated ferroptosis as a potentially targetable mechanism for cancer immunotherapy and supports PSD as a proof-of-concept lead compound and preclinical pharmacological probe for investigating combination strategies with ICT.