Successful immunogenic apoptosis in experimental cancer therapy depends on the induction of strong host anti-tumor responses. Given that tumors are often resistant to apoptosis, it is important to identify alternative molecular mechanisms that elicit immunogenic cell death. We have developed a genetic model in which direct dimerization of FADD combined with inducible expression of RIPK3 promotes necroptosis. We report that necroptotic cancer cells release damage-associated molecular patterns and promote maturation of dendritic cells, the cross-priming of cytotoxic T cells, and the production of IFN-γ in response to tumor antigen stimulation. Using both FADD-dependent and FADD-independent RIPK3 induction systems, we demonstrate the efficient vaccination potential of immunogenic necroptotic cells. Our study broadens the current concept of immunogenic cell death and opens doors for the development of new strategies in cancer therapy.

TNF is a master proinflammatory cytokine whose pathogenic role in inflammatory disorders can, in certain conditions, be attributed to RIPK1 kinase-dependent cell death. Survival, however, is the default response of most cells to TNF stimulation, indicating that cell demise is normally actively repressed and that specific checkpoints must be turned off for cell death to proceed. We identified RIPK1 as a direct substrate of MK2 in the TNFR1 signalling pathway. Phosphorylation of RIPK1 by MK2 limits cytosolic activation of RIPK1 and the subsequent assembly of the death complex that drives RIPK1 kinase-dependent apoptosis and necroptosis. In line with these in vitro findings, MK2 inactivation greatly sensitizes mice to the cytotoxic effects of TNF in an acute model of sterile shock caused by RIPK1-dependent cell death. In conclusion, we identified MK2-mediated RIPK1 phosphorylation as an important molecular mechanism limiting the sensitivity of the cells to the cytotoxic effects of TNF.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes a state of cellular stress known as ER stress. The cells respond to ER stress by activating the unfolded protein response (UPR), a signaling network emerging from the ER-anchored receptors IRE1α, PERK and ATF6. The UPR aims at restoring ER protein-folding homeostasis, but turns into a toxic signal when the stress is too severe or prolonged. Recent studies have demonstrated links between the UPR and inflammation. Consequently, small molecule inhibitors of IRE1α and PERK have become attractive tools for the potential therapeutic manipulation of the UPR in inflammatory conditions. TNF is a master pro-inflammatory cytokine that drives inflammation either directly by promoting gene activation, or indirectly by inducing RIPK1 kinase-dependent cell death, in the form of apoptosis or necroptosis. To evaluate the potential contribution of the UPR to TNF-induced cell death, we tested the effects of two commonly used PERK inhibitors, GSK2606414 and GSK2656157. Surprisingly, we observed that both compounds completely repressed TNF-mediated RIPK1 kinase-dependent death, but found that this effect was independent of PERK inactivation. Indeed, these two compounds turned out to be direct RIPK1 inhibitors, with comparable potency to the recently developed RIPK1 inhibitor GSK'963 (about 100 times more potent than NEC-1s). Importantly, these compounds completely inhibited TNF-mediated RIPK1-dependent cell death at a concentration that did not affect PERK activity in cells. In vivo, GSK2656157 administration protected mice from lethal doses of TNF independently of PERK inhibition and as efficiently as GSK'963. Together, our results not only report on new and very potent RIPK1 inhibitors but also highlight the risk of misinterpretation when using these two PERK inhibitors in the context of ER stress, cell death and inflammation.