High-risk neuroblastoma is a devastating malignancy with very limited therapeutic options. Here, we identify withaferin A (WA) as a natural ferroptosis-inducing agent in neuroblastoma, which acts through a novel double-edged mechanism. WA dose-dependently either activates the nuclear factor-like 2 pathway through targeting of Kelch-like ECH-associated protein 1 (noncanonical ferroptosis induction) or inactivates glutathione peroxidase 4 (canonical ferroptosis induction). Noncanonical ferroptosis induction is characterized by an increase in intracellular labile Fe(II) upon excessive activation of heme oxygenase-1, which is sufficient to induce ferroptosis. This double-edged mechanism might explain the superior efficacy of WA as compared with etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells, and in suppressing the growth and relapse rate of neuroblastoma xenografts. Nano-targeting of WA allows systemic application and suppressed tumor growth due to an enhanced accumulation at the tumor site. Collectively, our data propose a novel therapeutic strategy to efficiently kill cancer cells by ferroptosis.

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.

ADAR1 interaction with Z-RNA promotes editing of endogenous double-stranded RNA and prevents MDA5-dependent immune activation Graphical abstract Highlights d Z-RNA interaction with the Za domain of ADAR1 promotes editing of self-dsRNA d Mutation of the Za domain of ADAR1 triggers an MDA5/ MAVS-dependent IFN-I response d Homozygous ADAR1 Za domain mutant mice develop a spontaneous IFN-I signature d Hemizygous expression of Za domain mutant ADAR1 causes MAVS-dependent lethality

Immunogenic cell death significantly contributes to the success of anti-cancer therapies, but immunogenicity of different cell death modalities widely varies. Ferroptosis, a form of cell death that is characterized by iron accumulation and lipid peroxidation, has not yet been fully evaluated from this perspective. Here we present an inducible model of ferroptosis, distinguishing three phases in the process—‘initial’ associated with lipid peroxidation, ‘intermediate’ correlated with ATP release and ‘terminal’ recognized by HMGB1 release and loss of plasma membrane integrity—that serves as tool to study immune cell responses to ferroptotic cancer cells. Co-culturing ferroptotic cancer cells with dendritic cells (DC), reveals that ‘initial’ ferroptotic cells decrease maturation of DC, are poorly engulfed, and dampen antigen cross-presentation. DC loaded with ferroptotic, in contrast to necroptotic, cancer cells fail to protect against tumor growth. Adding ferroptotic cancer cells to immunogenic apoptotic cells dramatically reduces their prophylactic vaccination potential. Our study thus shows that ferroptosis negatively impacts antigen presenting cells and hence the adaptive immune response, which might hinder therapeutic applications of ferroptosis induction.

Lipid peroxidation (LPO) drives ferroptosis execution. However, LPO has been shown to contribute also to other modes of regulated cell death (RCD). To clarify the role of LPO in different modes of RCD, we studied in a comprehensive approach the differential involvement of reactive oxygen species (ROS), phospholipid peroxidation products, and lipid ROS flux in the major prototype modes of RCD viz. apoptosis, necroptosis, ferroptosis, and pyroptosis. LC-MS oxidative lipidomics revealed robust peroxidation of three classes of phospholipids during ferroptosis with quantitative predominance of phosphatidylethanolamine species. Incomparably lower amounts of phospholipid peroxidation products were found in any of the other modes of RCD. Nonetheless, a strong increase in lipid ROS levels was detected in non-canonical pyroptosis, but only during cell membrane rupture. In contrast to ferroptosis, lipid ROS apparently was not involved in non-canonical pyroptosis execution nor in the release of IL-1β and IL-18, while clear dependency on CASP11 and GSDMD was observed. Our data demonstrate that ferroptosis is the only mode of RCD that depends on excessive phospholipid peroxidation for its cytotoxicity. In addition, our results also highlight the importance of performing kinetics and using different methods to monitor the occurrence of LPO. This should open the discussion on the implication of particular LPO events in relation to different modes of RCD.

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Radiotherapy is commonly used as a cytotoxic treatment of a wide variety of tumors. Interestingly, few case reports underlined its potential to induce immune-mediated abscopal effects, resulting in regression of metastases, distant from the irradiated site. These observations are rare, and apparently depend on the dose used, suggesting that dose-related cellular responses may be involved in the distant immunogenic responses. Ionizing radiation (IR) has been reported to elicit immunogenic apoptosis, necroptosis, mitotic catastrophe, and senescence. In order to link a cellular outcome with a particular dose of irradiation, we performed a systematic study in a panel of cell lines on the cellular responses at different doses of X-rays. Remarkably, we observed that all cell lines tested responded in a similar fashion to IR with characteristics of mitotic catastrophe, senescence, lipid peroxidation, and caspase activity. Iron chelators (but not Ferrostatin-1 or vitamin E) could prevent the formation of lipid peroxides and cell death induced by IR, suggesting a crucial role of iron-dependent cell death during high-dose irradiation. We also show that in K-Ras-mutated cells, IR can induce morphological features reminiscent of methuosis, a cell death modality that has been recently described following H-Ras or K-Ras mutation overexpression.

The most common cause of death in the intensive care unit (ICU) is the development of multiorgan dysfunction syndrome (MODS). Besides life-supporting treatments, no cure exists, and its mechanisms are still poorly understood. Catalytic iron is associated with ICU mortality and is known to cause free radical-mediated cellular toxicity. It is thought to induce excessive lipid peroxidation, the main characteristic of an iron-dependent type of cell death conceptualized as ferroptosis. Here we show that the severity of multiorgan dysfunction and the probability of death are indeed associated with plasma catalytic iron and lipid peroxidation. Transgenic approaches underscore the role of ferroptosis in iron-induced multiorgan dysfunction. Blocking lipid peroxidation with our highly soluble ferrostatin-analogue protects mice from injury and death in experimental non-septic multiorgan dysfunction, but not in sepsis-induced multiorgan dysfunction. The limitations of the experimental mice models to mimic the complexity of clinical MODS warrant further preclinical testing. In conclusion, our data suggest ferroptosis targeting as possible treatment option for a stratifiable subset of MODS patients.