Article | Published:

eIF2α phosphorylation is pathognomonic for immunogenic cell death


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Edited by S. Fulda


  1. 1.

    Sharma P, Allison JP. Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential. Cell. 2015;161:205–14.

  2. 2.

    Palucka AK, Coussens LM. The basis of oncoimmunology. Cell. 2016;164:1233–47.

  3. 3.

    Pitt JM, Vetizou M, Daillere R, Roberti MP, Yamazaki T, Routy B, et al. Resistance mechanisms to immune-checkpoint blockade in cancer: tumor-intrinsic and -extrinsic factors. Immunity. 2016;44:1255–69.

  4. 4.

    Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. 2017;168:707–23.

  5. 5.

    Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366:2443–54.

  6. 6.

    Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455–65.

  7. 7.

    Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P, Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer. 2012;12:860–75.

  8. 8.

    Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer therapy. Annu Rev Immunol. 2013;31:51–72.

  9. 9.

    Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer G. Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol. 2017;17:97–111.

  10. 10.

    Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007;13:54–61.

  11. 11.

    Michaud M, Martins I, Sukkurwala AQ, Adjemian S, Ma Y, Pellegatti P, et al. Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice. Science. 2011;334:1573–7.

  12. 12.

    Martins I, Wang Y, Michaud M, Ma Y, Sukkurwala AQ, Shen S, et al. Molecular mechanisms of ATP secretion during immunogenic cell death. Cell Death Differ. 2014;21:79–91.

  13. 13.

    Ma Y, Adjemian S, Mattarollo SR, Yamazaki T, Aymeric L, Yang H, et al. Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells. Immunity. 2013;38:729–41.

  14. 14.

    Ko A, Kanehisa A, Martins I, Senovilla L, Chargari C, Dugue D, et al. Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signalling. Cell death Differ. 2014;21:92–9.

  15. 15.

    Ma Y, Galluzzi L, Zitvogel L, Kroemer G, Autophagy and cellular immune responses. Immunity. 2013;39:211–27.

  16. 16.

    Senovilla L, Vitale I, Martins I, Tailler M, Pailleret C, Michaud M, et al. An immunosurveillance mechanism controls cancer cell ploidy. Science. 2012;337:1678–84.

  17. 17.

    Vacchelli E, Ma Y, Baracco EE, Sistigu A, Enot DP, Pietrocola F, et al. Chemotherapy-induced antitumor immunity requires formyl peptide receptor 1. Science. 2015;350:972–8.

  18. 18.

    Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer G. Immunological effects of conventional chemotherapy and targeted anticancer agents. Cancer Cell. 2015;28:690–714.

  19. 19.

    Pietrocola F, Pol J, Vacchelli E, Rao S, Enot DP, Baracco EE, et al. Caloric restriction mimetics enhance anticancer immunosurveillance. Cancer Cell. 2016;30:147–60.

  20. 20.

    Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, Chaput N, et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med. 2005;202:1691–701.

  21. 21.

    Pfirschke C, Engblom C, Rickelt S, Cortez-Retamozo V, Garris C, Pucci F, et al. Immunogenic chemotherapy sensitizes tumors to checkpoint blockade therapy. Immunity. 2016;44:343–54.

  22. 22.

    Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12:298–306.

  23. 23.

    Mlecnik B, Bindea G, Angell HK, Maby P, Angelova M, Tougeron D, et al. Integrative analyses of colorectal cancer show immunoscore is a stronger predictor of patient survival than microsatellite instability. Immunity. 2016;44:698–711.

  24. 24.

    Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med. 2007;13:1050–9.

  25. 25.

    Vacchelli E, Enot DP, Pietrocola F, Zitvogel L, Kroemer G. Impact of pattern recognition receptors on the prognosis of breast cancer patients undergoing adjuvant chemotherapy. Cancer Res. 2016;76:3122–6.

  26. 26.

    Tesniere A, Schlemmer F, Boige V, Kepp O, Martins I, Ghiringhelli F, et al. Immunogenic death of colon cancer cells treated with oxaliplatin. Oncogene. 2010;29:482–91.

  27. 27.

    Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G. Cell death assays for drug discovery. Nat Rev Drug Discov. 2011;10:221–37.

  28. 28.

    Menger L, Vacchelli E, Adjemian S, Martins I, Ma Y, Shen S, et al. Cardiac glycosides exert anticancer effects by inducing immunogenic cell death. Sci Transl Med. 2012;4:143ra99

  29. 29.

    Sukkurwala AQ, Adjemian S, Senovilla L, Michaud M, Spaggiari S, Vacchelli E, et al. Screening of novel immunogenic cell death inducers within the NCI mechanistic diversity set. Oncoimmunology. 2014;3:e28473

  30. 30.

    Fucikova J, Becht E, Iribarren K, Goc J, Remark R, Damotte D, et al. Calreticulin expression in human non-small cell lung cancers correlates with increased accumulation of antitumor immune cells and favorable prognosis. Cancer Res. 2016;76:1746–56.

  31. 31.

    Fucikova J, Truxova I, Hensler M, Becht E, Kasikova L, Moserova I, et al. Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients. Blood. 2016;128:3113–24.

  32. 32.

    Donnelly N, Gorman AM, Gupta S, Samali A. The eIF2alpha kinases: their structures and functions. Cell Mol Life Sci. 2013;70:3493–511.

  33. 33.

    Choy MS, Yusoff P, Lee IC, Newton JC, Goh CW, Page R, et al. Structural and Functional Analysis of the GADD34:PP1 eIF2alpha Phosphatase. Cell Rep. 2015;11:1885–91.

  34. 34.

    Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell. 2003;11:619–33.

  35. 35.

    Lu PD, Jousse C, Marciniak SJ, Zhang Y, Novoa I, Scheuner D, et al. Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2. EMBO J. 2004;23:169–79.

  36. 36.

    Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM. The integrated stress response. EMBO Rep. 2016;17:1374–95.

  37. 37.

    Wang M, Kaufman RJ. The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat Rev Cancer. 2014;14:581–97.

  38. 38.

    Bettigole SE, Glimcher LH. Endoplasmic reticulum stress in immunity. Annu Rev Immunol. 2015;33:107–38.

  39. 39.

    Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC, et al. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J. 2009;28:578–90.

  40. 40.

    Panas MD, Ivanov P, Anderson P. Mechanistic insights into mammalian stress granule dynamics. J Cell Biol. 2016;215:313–23.

  41. 41.

    Talloczy Z, Jiang W, Virgin HWt, Leib DA, Scheuner D, Kaufman RJ, et al. Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proc Natl Acad Sci USA. 2002;99:190–5.

  42. 42.

    Shen S, Niso-Santano M, Adjemian S, Takehara T, Malik SA, Minoux H, et al. Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity. Mol Cell. 2012;48:667–80.

  43. 43.

    Leavitt R, Schlesinger S, Kornfeld S. Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. J Virol. 1977;21:375–85.

  44. 44.

    Lytton J, Westlin M, Hanley MR. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J Biol Chem. 1991;266:17067–71.

  45. 45.

    Garg AD, Krysko DV, Verfaillie T, Kaczmarek A, Ferreira GB, Marysael T, et al. A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J. 2012;31:1062–79.

  46. 46.

    Bloy N, Sauvat A, Chaba K, Buque A, Humeau J, Bravo-San Pedro JM, et al. Morphometric analysis of immunoselection against hyperploid cancer cells. Oncotarget. 2015;6:41204–15.

  47. 47.

    Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell. 2000;6:1099–108.

  48. 48.

    Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol. 2012;13:89–102.

  49. 49.

    Kepp O, Galluzzi L, Giordanetto F, Tesniere A, Vitale I, Martins I, et al. Disruption of the PP1/GADD34 complex induces calreticulin exposure. Cell Cycle. 2009;8:3971–7.

  50. 50.

    Guo L, Chi Y, Xue J, Ma L, Shao Z, Wu J. Phosphorylated eIF2alpha predicts disease-free survival in triple-negative breast cancer patients. Sci Rep. 2017;7:44674.

  51. 51.

    Martins I, Kepp O, Schlemmer F, Adjemian S, Tailler M, Shen S, et al. Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress. Oncogene. 2011;30:1147–58.

  52. 52.

    van Galen P, Kreso A, Mbong N, Kent DG, Fitzmaurice T, Chambers JE, et al. The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress. Nature. 2014;510:268–72.

  53. 53.

    Scheuner D, Song B, McEwen E, Liu C, Laybutt R, Gillespie P, et al. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol Cell. 2001;7:1165–76.

Download references


We thank David Enot for his help in mathematical modeling and Gautier Stoll for his help in statistical analysis in hyperploid experiments, Prof. Dr. Claudio Hetz for help with the XPB1 reverse transcription PCR, Prof. Dr. John E. Dick for sharing the pSMALB-ATF4.5rep plasmid [52] and Dr. Silke Nock and Prof. Dr. Peter Walter for the U2OS GFP-ATF6 cell line. GK is supported by the Ligue contre le Cancer (équipe labelisée); Agence National de la Recherche (ANR) – Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Institut National du Cancer (INCa); Institut Universitaire de France; Fondation pour la Recherche Médicale (FDM20140630126 and FDM 40739); the European Commission (ArtForce); the European Research Council (ERC); the LeDucq Foundation; the LabEx Immuno-Oncology; the RHU Torino Lumière, the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); and the Paris Alliance of Cancer Research Institutes (PACRI). PL is supported by the Chinese Scholarship Council and LZ by la Ligue contre le cancer. LS is supported by Fondation ARC projects. JH owns a Fondation Philantropia’s fellowship. LG is supported by Fundação para a Ciência e a Tecnologia (FCT).

Author information

Conflict of interest

The authors declare that they have no conflict of interest.

Correspondence to Oliver Kepp or Guido Kroemer.

Electronic supplementary material

Figure S1. Original matrix of correlation

Figure S2. Protein translation

Figure S3. GRP78 but not both Calreticulin and PDIA3 increases during ER stress

Figure S4. Calreticulin and PDIA3 but not GRP78 translocate to the cell surface

Figure S5. Calreticulin, PDIA3 translocate to the cell surface

Figure S6.EIF2AK3/PERK is essential to eIF2α phosphorylation and calreticulin exposure

Figure S7. No eIF2α phosphorylation affects autophagy induction but not HMGB1 release

Figure S8. Validation of the predicting model

Figure S9. Calculation of a predicted ICD score

Table S1. Raw Data

Table S2. Raw Data

Table S3. Model Matrix Coefficients

Supplemental Figures and Tables

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1: Anthracyclines induce the phosphorylation of eIF2α yet fail to induce other markers of ER stress
Fig. 2: Differential stress patterns evoked by immunogenic cell death-inducing and autophagy-inducing drugs
Fig. 3: Partial split of ER stress responses in vivo induced by ICD drugs
Fig. 4: Correlation of cell stress and cell death parameters
Fig. 5: CALR exposure depends on eIF2α phosphorylation yet is independent of the ER stress
Fig. 6: Mutually exclusive induction of ER stress and calreticulin exposure
Fig. 7: Hyperploidy-associated signs of ER stress
Fig. 8: Chemical descriptors and immunogenic cell death prediction