eIF2α phosphorylation is pathognomonic for immunogenic cell death

Abstract

The phosphorylation of eIF2α is essential for the endoplasmic reticulum (ER) stress response, the formation of stress granules, as well as macroautophagy. Several successful anticancer chemotherapeutics have the property to induce immunogenic cell death (ICD), thereby causing anticancer immune responses. ICD is accompanied by the translocation of calreticulin (CALR) from the ER lumen to the plasma membrane, which facilitates the transfer of tumor-associated antigens to dendritic cells. Here we systematically investigated the capacity of anticancer chemotherapeutics to induce signs of ER stress. ICD inducers including anthracyclines and agents that provoke tetraploidization were highly efficient in enhancing the phosphorylation of eIF2α, yet failed to stimulate other signs of ER stress including the transcriptional activation of activating transcription factor 4 (ATF4), the alternative splicing of X-box binding protein 1 (XBP1s) mRNA and the proteolytic cleavage of activating transcription factor 6 (ATF6) both in vitro and in cancers established in mice. Systematic analyses of clinically used anticancer chemotherapeutics revealed that only eIF2α phosphorylation, but none of the other signs of ER stress, correlated with CALR exposure. eIF2α phosphorylation induced by mitoxantrone, a prototype ICD-inducing anthracyline, was mediated by eIF2α kinase-3 (EIF2AK3). Machine-learning approaches were used to determine the physicochemical properties of drugs that induce ICD, revealing that the sole ER stress response relevant to the algorithm is eIF2α phosphorylation with its downstream consequences CALR exposure, stress granule formation and autophagy induction. Importantly, this approach could reduce the complexity of compound libraries to identify ICD inducers based on their physicochemical and structural characteristics. In summary, it appears that eIF2α phosphorylation constitutes a pathognomonic characteristic of ICD.

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

“…Despite the phosphorylation of eIF2α, neither the activation of ATF4 nor that of DDIT3 was observed. Similar findings were recently reported for different ICD drugs reinforcing the notion of the phosphorylation of eIF2α as an important ICD hallmark (Bezu et al , ). In line with this observation, other ICD hallmarks (such as the exposure of CALR, the secretion of ATP, and the exodus of HMGB1) were detected in response to redaporfin‐PDT and, more importantly, cells killed in vitro by redaporfin‐PDT were able to vaccinate mice against rechallenge with live cancer cells.…”

“…eIF2α phosphorylation without the activation of ATF4 and DDIT3 was recently described as a hallmark of ICD (Bezu et al , ). Indeed, redaporfin‐based PDT induced other hallmarks of ICD such as plasma membrane calreticulin exposure, as well as the release of ATP and HMGB1 from cancer cells in vitro (Appendix Fig S4).…”

Linking cellular stress responses to systemic homeostasis

“…Accordingly, TERS-primed cancer cells grow more quickly in vivo than their control counterparts 62 . Some cell-deathpromoting chemotherapeutic agents such as anthracyclines and oxaliplatin also trigger a partial ER stress response in cancer cells, which manifests with PERKdependent eIF2α phosphorylation but not with ATF4, ATF6 and XBP1s activation 63 . Phosphorylated eIF2α favours the translocation of ER chaperones including calreticulin (CALR) and protein disulfide-isomerase A3 (PDIA3) from the ER lumen to the cell surface 64 , where they act as 'eat-me' signals to promote phagocytosis of dying cancer cells by dendritic cells and hence initiate anticancer immunity 17 .…”

“…However, in response to certain stimuli, including anthracyclines, oxaliplatin and hypofractionated irradiation, apoptosis can mediate robust immunostimulatory effects 17,173 . Such a form of immunogenic cell death (ICD) mechanistically relies on the activation of intracellular stress responses, including (but perhaps not limited to) a partial ER stress response (culminating in the exposure of pro-phagocytic signals; see above) 63 , autophagy (resulting in ATP release; see above) 110 and intracellular nucleic acid sensing (resulting in type I IFN release, see above) 25,174 . Interestingly, apoptotic ICD depends on CASP8 (REF.…”

Treatment of colon cancer with liver X receptor agonists induces immunogenic cell death

“…Furthermore, cytofluorometric analysis showed that T0901317 induced significant translocation of CRT to the cell surface in HCT116 cells ( Figure 1B). As CRT exposure occurs as a consequence of ER stress-induced phosphorylation of eIF2α, 17,18 we then examined expression levels of eIF2α and P-eIF2α after treatment with T0901317. As shown in Figure 1C, HCT116 cells treated with T0901317 increased expression of P-eIF2α.…”