Effect of RIPK3 on non-canonical PANoptosis to suppress anti-tumor immunity in lung cancer.

e14565 Background: Hypoxia and glucose deprivation are common features of solid malignancies and considered the causes of tumor necrosis, while the molecular mechanisms of tumor cell death and its impact on tumor progression are not yet clear. PANoptosis is a newly recognized programmed cell death pathway, characterized by the simultaneous activation of necroptosis, apoptosis and pyroptosis. The canonical PANoptosis is controlled by an ASC-dependent multimeric protein complex named the PANoptosome. Methods: Using immunoblot and immunofluorescent analysis, we described the RIPK3 expression patterns and detected the programmed cell death markers in lung cancer tissues. We built a RIPK32×Fv-Flag-expressing PKLC cell line and identified the key molecules involved in tumor cell death by using MS analysis of RIPK3-immunoprecipitate. These molecules were confirmed by CRISPR-Cas9 mediated gene knockout. Then we analyzed the secretome by 4D-diaXLMS of cell death conditioned medium and identified a variety of DAMPs and cytokines released by PANoptotic tumor cells. Last, the differences of tumor growth and the immune microenvironment between wild-type and Ripk3 KO PKLC-CDX were compared. Results: Hypoxia and glucose deprivation could induce over expression of RIPK3 and mediated PANoptosis of tumor cells. The oligomerized RIPK3 rapidly recruits MLKL and mediates MLKL phosphorylation to induce necroptosis. Further aggregated RIPK3 can recruit RIPK1 and assembles with FADD and Caspase-8, causing Caspase-8 auto-cleavage. Active Caspase-8 can directly cleave caspase-3 and GSDMD to induce apoptosis and pyroptosis. The PANoptosis we described is initiated by RIPK3 and independent of ASC, which we termed non-canonical PANoptosis. We also found that Caspase-3, as an executor of apoptosis, is able to cleave RIPK3, RIPK1 and truncated GSDMD, negatively regulating PANoptotic cell death. Finally, we identified a variety of DAMPs released by PANoptotic tumor cells, which facilitate neutrophil differentiation and activation, promoting tumor progression by suppressing anti-tumor immunity. Conclusions: Hypoxia and glucose deprivation induce non-canonical PANoptosis that is initiated by RIPK3 and independent of ASC. The PANoptotic tumor cells suppress anti-tumor immunity through releasing DAMPs and cytokines to mediate neutrophil differentiation and activation, which promote lung cancer progression.