The DAPK3-DCAF1 pathway regulates ZBP1 protein stability to orchestrate PANoptosis for ovarian cancer therapy

Ovarian cancer is characterized by an immunosuppressive "cold" tumor microenvironment, which poses a major challenge to effective therapy. Inducing immunogenic cell death through PANoptosis represents a promising strategy for remodeling the tumor microenvironment. Z-DNA binding protein 1 (ZBP1), an interferon (IFN)-stimulated gene, is a key sensor of Z-conformation nucleic acids (Z-NA) driving PANoptosis. While ZBP1 upregulation is traditionally attributed to transcriptional induction, its early non-transcriptional regulatory mechanisms remain elusive.

ZBP1 expression and its prognostic value were analyzed using public databases and clinical cohorts. APEX2 proximity labeling, PLA and Co-IP identified the E3 ubiquitin ligase regulating ZBP1. The IFN-mediated ZBP1 post-translational modification pathway was delineated utilizing PLA, Co-IP, in vitro phosphorylation, mutagenesis assays, an intestine-specific conditional knockout model. DCAF1^-/- ovarian cancer cells and immunocompetent ID8 peritoneal models were generated to evaluate tumor growth and cell death. The therapeutic efficacy of combining the DCAF1 inhibitor (B32B3) with the Z-NA inducer (CBL0137) was assessed in an immunocompetent ID8 murine peritoneal tumor model and two chemoresistant patient-derived xenograft (PDX) models.

ZBP1 is significantly downregulated in ovarian cancer, whereas its elevated expression predicts a favorable prognosis and correlates with high IFN responsiveness. Type I IFN triggers a rapid accumulation of ZBP1 protein prior to its transcriptional upregulation. Mechanistically, we identified the E3 ligase substrate receptor DCAF1 as a negative regulator that targets ZBP1 for proteasomal degradation. Interferon signaling activates the kinase DAPK3, which phosphorylates DCAF1 at S1328. This phosphorylation event compromises the assembly of the CRL4^DCAF1 complex, thereby abrogating DCAF1-mediated degradation of ZBP1. In ovarian cancer models, genetic ablation of DCAF1 restored ZBP1 levels and significantly restrained tumor progression. Therapeutically, combining B32B3 with CBL0137 elevated intracellular Z-NA and stabilized ZBP1, driving PANoptosis and suppressing ovarian tumor growth across.

Our study identifies the IFN-DAPK3-DCAF1 pathway as a critical post-translational mechanism that ensures rapid ZBP1 stabilization. This highlights a fundamental strategy to bypass transcriptional latency for the rapid activation of immune responses, offering a strong clinical rationale to harness this pathway to induce ZBP1-dependent PANoptosis for the treatment of refractory tumors.