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Essential role for Bim in mediating the apoptotic and antitumor activities of immunotoxins

Oncogene volume 36, pages 4953–4962 (2017)Cite this article

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Abstract

Protein synthesis is crucial for regulating cell homeostasis and, when unrestricted, it can lead to tumorigenesis. Immunotoxins derived from Pseudomonas exotoxin are antibody–toxin fusion proteins that inhibit protein synthesis of mammalian cells via ADP-ribosylation of the eukaryotic elongation factor-2. Here we investigate the role of the Bcl-2 family proteins in the response of cancer cells to immunotoxin challenge. Besides the well-known reduction of the prosurvival Bcl-2 family member, Mcl-1, following inhibition of protein synthesis, we show for the first time that immunotoxins also reduce the levels of selected proapoptotic BH-3-only proteins. Among these, only Bim protein levels correlated with the ability of immunotoxins to induce an apoptotic response. To support our findings, we verified that a Bim knockout completely abolished immunotoxin-mediated apoptosis. Further, mice bearing either wild-type or Bid knockout tumors responded to immunotoxin treatment with a decrease in growth kinetics, whereas mice engrafted with Bim knockout tumors showed no reduction in tumor size or prolongation of survival following immunotoxin treatment. From these results, we conclude that Bim expression is a major susceptibility factor for tumor cell death and, as such, constitutes a potential biomarker that could be evaluated before immunotoxin treatment. In support of this hypothesis, clinically, we analyzed patient cells before immunotoxin treatment and report that samples of hairy cell leukemia with high levels of Bim protein responded with a greater decrease in leukemic cell count compared with those samples expressing a low level of Bim.

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References

  1. Adams JM, Cory S . Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol 2007; 19: 488–496.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR . The BCL-2 family reunion. Mol Cell 2010; 37: 299–310.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hata AN, Engelman JA, Faber AC . The BCL2 family: key mediators of the apoptotic response to targeted anticancer therapeutics. Cancer Discov 2015; 5: 475–487.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Weldon JE, Pastan I . A guide to taming a toxin—recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J 2011; 278: 4683–4700.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Antignani A, Fitzgerald D . Immunotoxins: the role of the toxin. Toxins (Basel) 2013; 5: 1486–1502.

    Article  CAS  Google Scholar 

  6. FitzGerald DJ, Wayne AS, Kreitman RJ, Pastan I . Treatment of hematologic malignancies with immunotoxins and antibody-drug conjugates. Cancer Res 2011; 71: 6300–6309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wayne AS, Fitzgerald DJ, Kreitman RJ, Pastan I . Immunotoxins for leukemia. Blood 2014; 123: 2470–2477.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Alewine C, Hassan R, Pastan I . Advances in anticancer immunotoxin therapy. Oncologist 2015; 20: 176–185.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Antignani A, Sarnovsky R, FitzGerald DJ . ABT-737 promotes the dislocation of ER luminal proteins to the cytosol, including Pseudomonas exotoxin. Mol Cancer Ther 2014; 13: 1655–1663.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Andersson Y, Juell S, Fodstad O . Downregulation of the antiapoptotic MCL-1 protein and apoptosis in MA-11 breast cancer cells induced by an anti-epidermal growth factor receptor-Pseudomonas exotoxin a immunotoxin. Int J Cancer 2004; 112: 475–483.

    Article  CAS  PubMed  Google Scholar 

  11. Traini R, Ben-Josef G, Pastrana DV, Moskatel E, Sharma AK, Antignani A et al. ABT-737 overcomes resistance to immunotoxin-mediated apoptosis and enhances the delivery of Pseudomonas exotoxin-based proteins to the cell cytosol. Mol Cancer Ther 2010; 9: 2007–2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hollevoet K, Antignani A, Fitzgerald DJ, Pastan I . Combining the antimesothelin immunotoxin SS1P with the BH3-mimetic ABT-737 induces cell death in SS1P-resistant pancreatic cancer cells. J Immunother 2014; 37: 8–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lindqvist LM, Vikstrom I, Chambers JM, McArthur K, Ann Anderson M, Henley KJ et al. Translation inhibitors induce cell death by multiple mechanisms and Mcl-1 reduction is only a minor contributor. Cell Death Dis 2012; 3: e409.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Willis SN, Fletcher JI, Kaufmann T, van Delft MF, Chen L, Czabotar PE et al. Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 2007; 315: 856–859.

    Article  CAS  PubMed  Google Scholar 

  15. Ren D, Tu HC, Kim H, Wang GX, Bean GR, Takeuchi O et al. BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program. Science 2010; 330: 1390–1393.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kreitman RJ, Tallman MS, Robak T, Coutre S, Wilson WH, Stetler-Stevenson M et al. Phase I trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. J Clin Oncol 2012; 30: 1822–1828.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kreitman RJ, Pastan I . Immunoconjugates in the management of hairy cell leukemia. Best Pract Res Clin Haematol 2015; 28: 236–245.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Salvatore G, Nagata S, Billaud M, Santoro M, Vecchio G, Pastan I . Generation and characterization of novel monoclonal antibodies to the Ret receptor tyrosine kinase. Biochem Biophys Res Commun 2002; 294: 813–817.

    Article  CAS  PubMed  Google Scholar 

  19. Antignani A, Mathews Griner L, Guha R, Simon N, Pasetto M, Keller J et al. Chemical screens identify drugs that enhance or mitigate cellular responses to antibody-toxin fusion proteins. PLoS ONE 2016; 11: e0161415.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 2005; 17: 393–403.

    Article  CAS  PubMed  Google Scholar 

  21. Buchner J, Pastan I, Brinkmann U . A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal Biochem 1992; 205: 263–270.

    Article  CAS  PubMed  Google Scholar 

  22. Aubrey BJ, Kelly GL, Kueh AJ, Brennan MS, O'Connor L, Milla L et al. An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo. Cell Rep 2015; 10: 1422–1432.

    Article  CAS  PubMed  Google Scholar 

  23. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72: 8463–8471.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 2003; 9: 493–501.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank R Anderson and W Welch for the gift of reagents and Jianan Gong and Stephen Wilcox for assistance with sequence validation of CRISPR/Cas9 knockout cell lines. We thank E Arons for advice on the RT–PCR. This work was funded by the intramural programs of the National Cancer Institute, NIH and is supported by fellowships, and grants from the Australian National Health and Medical Research Council (research fellowships to DCSH; project grant 1057742 (DCSH); program grant 1016701; and Independent Research Institutes Infrastructure Support Scheme (grant 9000220)), the Cancer Council Victoria (grant-in-aid to DCSH), the Leukemia and Lymphoma Society (Specialized Centers of Research grants 7001-13), the Australian Cancer Research Foundation, a Victorian State Government Operational Infrastructure Support grant.

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Authors and Affiliations

  1. Laboratory of Molecular Biology, CCR, NCI, National Institutes of Health, Bethesda, MD, USA

    A Antignani, N Simon, R J Kreitman & D J FitzGerald

  2. Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia

    D Segal & D Huang

  3. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia

    D Segal

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  1. A Antignani
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  2. D Segal
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  3. N Simon
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Correspondence to A Antignani or D J FitzGerald.

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Antignani, A., Segal, D., Simon, N. et al. Essential role for Bim in mediating the apoptotic and antitumor activities of immunotoxins. Oncogene 36, 4953–4962 (2017). https://doi.org/10.1038/onc.2017.111

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