DAPK3-DCAF1 pathway regulates ZBP1 protein stability to orchestrate innate immune responses and PANoptosis

Background: Z-DNA binding protein 1 (ZBP1) is an interferons (IFN)-stimulated cytosolic sensor of Z-nucleic acids (Z-NA) that regulates PANoptosis (pyroptosis, apoptosis, and/or necroptosis) and thereby drives potent anti-tumor immune responses. However, the mechanisms controlling ZBP1 protein stability in tumors-and whether they can be therapeutically manipulated to induce ICD-remain unclear. We sought to define regulatory pathways that govern ZBP1 abundance in ovarian cancer and to test strategies that exploit ZBP1 activation for antitumor therapy. Methods & Results: Here we demonstrate that the reduced ZBP1 expression in ovarian cancer and correlated with poorer survival. Type I IFN (IFN-beta) rapidly increased ZBP1 levels by inhibiting its proteasome-dependent ubiquitination. Proximity labeling and biochemical assays identified DCAF1 as a key interactor that mediates ZBP1 ubiquitination and degradation. Furthermore, IP-MS and LC-MS/MS identified DAPK3 as an IFN-induced DCAF1 kinase, which phosphorylates DCAF1-pS1328 to promote its turnover, thereby stabilizing ZBP1. Genetic ablation of DCAF1 impaired tumor cell proliferation in vitro , reduced tumor burden and promoted anti-tumor immunity in vivo . Pharmacological targeting of this pathway using the DCAF1 inhibitor B32B3 and-when combined with the Z-NA activator CBL0137-effectively reactivated ZBP1 signaling, promoted PANoptosis, and enhanced antitumor immunity, thereby suppressing tumor growth in two chemotherapy-resistant ovarian cancer patient-derived xenograft (PDX) models and providing a promising therapeutic strategy for refractory malignancies. Conclusion: Overall, this study delineates a novel DAPK3-DCAF1 signaling axis that governs ZBP1 stability, unveiling an integrated regulatory framework that connects interferon signaling, post-translational modification, and immunogenic cell death. Combining DCAF1 blockade with Z-NA activation elicits ZBP1-mediated PANoptosis and augments anti-tumor immunity, representing a promising therapeutic approach for refractory ovarian cancer.