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Chemical genetics identify eIF2α kinase heme-regulated inhibitor as an anticancer target

Nature Chemical Biology volume 7, pages 610–616 (2011)Cite this article

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Abstract

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2–GTP–tRNAiMet translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2–GTP–tRNAiMet ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N′-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N′-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2α and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N′-diarylureas are potent and specific tools for studying the role of eIF2–GTP–tRNAiMet ternary complex in the pathobiology of human disorders.

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Figure 1: Identification and validation of the N,N′-diarylureas as modifiers of the ternary complex abundance.
Figure 2: N,N′-diarylureas reduce the abundance of the ternary complex by causing phosphorylation of eIF2α.
Figure 3: The N,N′-diarylureas specifically activate HRI kinase.
Figure 4: Phosphorylation of eIF2α by HRI mediates inhibition of cancer cell proliferation by N,N′-diarylureas.
Figure 5: The in vitro and in vivo anticancer activity of N,N′-diarylureas.

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References

  1. Marshall, L., Kenneth, N.S. & White, R.J. Elevated tRNA(iMet) synthesis can drive cell proliferation and oncogenic transformation. Cell 133, 78–89 (2008).

    Article  CAS  Google Scholar 

  2. Ranganathan, A.C., Ojha, S., Kourtidis, A., Conklin, D.S. & Aguirre-Ghiso, J.A. Dual function of pancreatic endoplasmic reticulum kinase in tumor cell growth arrest and survival. Cancer Res. 68, 3260–3268 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  3. Harding, H.P. et al. Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development. Proc. Natl. Acad. Sci. USA 106, 1832–1837 (2009).

    Article  CAS  Google Scholar 

  4. Heaney, J.D., Michelson, M.V., Youngren, K.K., Lam, M.Y. & Nadeau, J.H. Deletion of eIF2beta suppresses testicular cancer incidence and causes recessive lethality in agouti-yellow mice. Hum. Mol. Genet. 18, 1395–1404 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  5. Skalnikova, H. et al. Protein signaling pathways in differentiation of neural stem cells. Proteomics 8, 4547–4559 (2008).

    Article  CAS  Google Scholar 

  6. Asano, K., Clayton, J., Shalev, A. & Hinnebusch, A.G. A multifactor complex of eukaryotic initiation factors, eIF1, eIF2, eIF3, eIF5, and initiator tRNA(Met) is an important translation initiation intermediate in vivo. Genes Dev. 14, 2534–2546 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  7. Algire, M.A., Maag, D. & Lorsch, J.R. Pi release from eIF2, not GTP hydrolysis, is the step controlled by start-site selection during eukaryotic translation initiation. Mol. Cell 20, 251–262 (2005).

    Article  CAS  Google Scholar 

  8. Prostko, C.R., Dholakia, J.N., Brostrom, M.A. & Brostrom, C.O. Activation of the double-stranded RNA-regulated protein kinase by depletion of endoplasmic reticular calcium stores. J. Biol. Chem. 270, 6211–6215 (1995).

    Article  CAS  Google Scholar 

  9. Srivastava, S.P., Davies, M.V. & Kaufman, R.J. Calcium depletion from the endoplasmic reticulum activates the double-stranded RNA-dependent protein kinase (PKR) to inhibit protein synthesis. J. Biol. Chem. 270, 16619–16624 (1995).

    Article  CAS  Google Scholar 

  10. Aktas, H. et al. Depletion of intracellular Ca2+ stores, phosphorylation of eIF2alpha, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole. Proc. Natl. Acad. Sci. USA 95, 8280–8285 (1998).

    Article  CAS  Google Scholar 

  11. Donzé, O., Jagus, R., Koromilas, A.E., Hershey, J.W. & Sonenberg, N. Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J. 14, 3828–3834 (1995).

    Article  PubMed Central  Google Scholar 

  12. Rosenwald, I.B., Hutzler, M.J., Wang, S., Savas, L. & Fraire, A.E. Expression of eukaryotic translation initiation factors 4E and 2alpha is increased frequently in bronchioloalveolar but not in squamous cell carcinomas of the lung. Cancer 92, 2164–2171 (2001).

    Article  CAS  Google Scholar 

  13. Abraham, N. et al. The murine PKR tumor suppressor gene is rearranged in a lymphocytic leukemia. Exp. Cell Res. 244, 394–404 (1998).

    Article  CAS  Google Scholar 

  14. Rosenwald, I.B. et al. Expression of the translation initiation factors eIF-4E and eIF-2* is frequently increased in neoplastic cells of Hodgkin lymphoma. Hum. Pathol. 39, 910–916 (2008).

    Article  CAS  Google Scholar 

  15. Rosenwald, I.B., Wang, S., Savas, L., Woda, B. & Pullman, J. Expression of translation initiation factor eIF-2alpha is increased in benign and malignant melanocytic and colonic epithelial neoplasms. Cancer 98, 1080–1088 (2003).

    Article  CAS  Google Scholar 

  16. Wek, R.C., Jiang, H.Y. & Anthony, T.G. Coping with stress: eIF2 kinases and translational control. Biochem. Soc. Trans. 34, 7–11 (2006).

    Article  CAS  Google Scholar 

  17. Chen, J.J. Regulation of protein synthesis by the heme-regulated eIF2alpha kinase: relevance to anemias. Blood 109, 2693–2699 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Chen, J.J. Heme-regulated elF-2a kinase. in Translational Control of Gene Expression (eds. Sonenberg, N. et al.) 529–546 (Cold Spring Harbor Laboratory Press, New York, 2000).

  19. Han, A.P., Fleming, M.D. & Chen, J.J. Heme-regulated eIF2alpha kinase modifies the phenotypic severity of murine models of erythropoietic protoporphyria and beta-thalassemia. J. Clin. Invest. 115, 1562–1570 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  20. Harding, H.P., Zhang, Y., Bertolotti, A., Zeng, H. & Ron, D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5, 897–904 (2000).

    Article  CAS  Google Scholar 

  21. Marbach, I., Licht, R., Frohnmeyer, H. & Engelberg, D. Gcn2 mediates Gcn4 activation in response to glucose stimulation or UV radiation not via GCN4 translation. J. Biol. Chem. 276, 16944–16951 (2001).

    Article  CAS  Google Scholar 

  22. Su, Q. et al. Modulation of the eukaryotic initiation factor 2 alpha-subunit kinase PERK by tyrosine phosphorylation. J. Biol. Chem. 283, 469–475 (2008).

    Article  CAS  Google Scholar 

  23. Biason-Lauber, A., Lang-Muritano, M., Vaccaro, T. & Schoenle, E.J. Loss of kinase activity in a patient with Wolcott-Rallison syndrome caused by a novel mutation in the EIF2AK3 gene. Diabetes 51, 2301–2305 (2002).

    Article  CAS  Google Scholar 

  24. Harding, H.P. et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099–1108 (2000).

    Article  CAS  Google Scholar 

  25. Lu, P.D., Harding, H.P. & Ron, D. Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J. Cell Biol. 167, 27–33 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  26. Vattem, K.M. & Wek, R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 11269–11274 (2004).

    Article  CAS  Google Scholar 

  27. Ziegeler, G. et al. Embryonic lethal abnormal vision-like HuR-dependent mRNA stability regulates post-transcriptional expression of cyclin-dependent kinase inhibitor p27Kip1. J. Biol. Chem. 285, 15408–15419 (2010).

    Article  CAS  PubMed Central  Google Scholar 

  28. Brewer, J.W. & Diehl, J.A. PERK mediates cell-cycle exit during the mammalian unfolded protein response. Proc. Natl. Acad. Sci. USA 97, 12625–12630 (2000).

    Article  CAS  Google Scholar 

  29. Lomenick, B. et al. Target identification using drug affinity responsive target stability (DARTS). Proc. Natl. Acad. Sci. USA 106, 21984–21989 (2009).

    Article  CAS  Google Scholar 

  30. Moerke, N.J. et al. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell 128, 257–267 (2007).

    Article  CAS  Google Scholar 

  31. Lu, L., Han, A.P. & Chen, J.J. Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol. Cell. Biol. 21, 7971–7980 (2001).

    Article  CAS  PubMed Central  Google Scholar 

  32. Scheuner, D. et al. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol. Cell 7, 1165–1176 (2001).

    Article  CAS  Google Scholar 

  33. Dever, T.E. Gene-specific regulation by general translation factors. Cell 108, 545–556 (2002).

    Article  CAS  Google Scholar 

  34. Koromilas, A.E. et al. The interferon-inducible protein kinase PKR modulates the transcriptional activation of immunoglobulin kappa gene. J. Biol. Chem. 270, 25426–25434 (1995).

    Article  CAS  Google Scholar 

  35. Boyce, M. et al. A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death. Cell Death Differ. 15, 589–599 (2008).

    Article  CAS  Google Scholar 

  36. Boyce, M. et al. A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science 307, 935–939 (2005).

    Article  CAS  Google Scholar 

  37. Harding, H.P. et al. Diabetes mellitus and exocrine pancreatic dysfunction in perk−/− mice reveals a role for translational control in secretory cell survival. Mol. Cell 7, 1153–1163 (2001).

    Article  CAS  Google Scholar 

  38. Araki, E., Oyadomari, S. & Mori, M. Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus. Exp. Biol. Med. (Maywood) 228, 1213–1217 (2003).

    Article  CAS  Google Scholar 

  39. Katayama, T. et al. Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response. Nat. Cell Biol. 1, 479–485 (1999).

    Article  CAS  Google Scholar 

  40. Xu, C., Bailly-Maitre, B. & Reed, J.C. Endoplasmic reticulum stress: cell life and death decisions. J. Clin. Invest. 115, 2656–2664 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  41. Palakurthi, S.S., Aktas, H., Grubissich, L.M., Mortensen, R.M. & Halperin, J.A. Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor gamma and mediated by inhibition of translation initiation. Cancer Res. 61, 6213–6218 (2001).

    CAS  PubMed  Google Scholar 

  42. Aktas, H., Cai, H. & Cooper, G.M. Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1. Mol. Cell. Biol. 17, 3850–3857 (1997).

    Article  CAS  PubMed Central  Google Scholar 

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Acknowledgements

We thank D.C. Tosteson (Harvard Medical School), mentor and role model to authors, for encouragement; J.J. Chen for providing the HRI expression plasmid; and M. Tosteson for critical comments on the manuscript. This work was supported in part by NIH grant NCDDG 5 U19 CA87427 to J.A.H., Susan B. Komen Foundation for Cancer Research grant BCTR0707713, US Department of Defense grant no. W81ZWH-05-1-0096 and US National Institutes of Health grant no. R21AG032546 to B.H.A.

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Authors and Affiliations

  1. Laboratory for Translational Research, Harvard Medical School, Boston, Massachusetts, USA

    Ting Chen, Duygu Ozel, Yuan Qiao, Fred Harbinski, Limo Chen, Séverine Denoyelle, Michael Chorev, Jose A Halperin & Bertal H Aktas

  2. Massachusetts General Hospital, Boston, Massachusetts, USA

    Xiaoying He, Nela Zvereva & Jeffrey G Supko

  3. Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA

    Michael Chorev, Jose A Halperin & Bertal H Aktas

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Contributions

T.C. characterized hits compounds, carried out experiments to determine the mechanism of action of hit compounds, analyzed the data and contributed to writing the paper; D.O. generated the screening assay and helped initial compound characterization; Y.Q. generated and characterized PC-3 cell lines; F.H. carried out screening campaign; L.C. carried out cell growth experiments; S.D. carried out small scale resynthesis of compounds; X.H., N.Z. and J.G.S. developed and validated an LC-MS method and analyzed mouse plasma samples; M.C. supervised synthesis and contributed to writing the paper; J.A.H. discussed the data and contributed to writing the manuscript; B.H.A. conceived, designed and supervised all aspects of research, analyzed the data and wrote the manuscript.

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Correspondence to Bertal H Aktas.

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Supplementary Methods and Supplementary Results (PDF 2473 kb)

Supplementary Dataset 1

Confirmation of initial hits with the ternary complex assay. Initial hits were confirmed in the ternary complex assay by testing in triplicate at 10 and 5 or 2.5 μM concentrations. Chemical structures can be obtained by entering the NSC number at http://dtp.nci.nih.gov/dtpstandard/ChemData/index.jsp. (XLS 78 kb)

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Chen, T., Ozel, D., Qiao, Y. et al. Chemical genetics identify eIF2α kinase heme-regulated inhibitor as an anticancer target. Nat Chem Biol 7, 610–616 (2011). https://doi.org/10.1038/nchembio.613

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