GDF15 regulates necroptotic cell death through direct interaction with RIPK3

ABSTRACT Enhancing the immunogenicity of tumor cells is a major objective in cancer therapy, particularly for tumors with low immune cell infiltration scores. Inducing immunogenic forms of programmed cell death (PCD) offers a promising strategy to strengthen anti-tumor immunity and improve therapeutic outcomes. Necroptosis, a highly inflammatory form of regulated cell death triggered by TNF signaling, can elicit robust immune activation. However, its regulation in tumor cells remains incompletely understood, limiting its therapeutic exploitation. To investigate the protein-protein interactions that govern necroptotic cell death in tumor cells, we established a co-Immunoprecipitation - Mass Spectrometry (coIP-MS) workflow using RIPK3, the central effector kinase driving necroptosis in TNF-induced signaling, as bait. This unbiased proteomic approach enables the identification of candidate regulators directly associated with the necrosome complex components under active necroptotic conditions. Among identified candidates, TCOF1 and GDF15 emerged as previously unrecognized modulators, with functional knockout of either gene markedly enhancing necroptotic cell death in tumor cells. Reciprocal IP experiments confirmed a direct interaction between GDF15 and RIPK3, supporting its mechanistic role as a negative regulator that suppresses necroptotic signaling. Thus, our findings extend the function of GDF15 beyond its established role in inflammation, uncovering an additional layer of regulation at the level of cell-intrinsic death signaling. Collectively, our findings position GDF15 as a RIPK3-interacting “brake” on necroptotic cell death and highlight TCOF1 as an additional inhibitory node. Our study underscores the potential of targeting necroptosis-suppressive mechanisms to influence PCD outcomes in tumors and demonstrates the power of coIP-MS for mapping TNF-induced interactions to reveal actionable molecular targets for tumor sensitization.