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Multiple myeloma gammopathies

A genome-scale CRISPR-Cas9 screening in myeloma cells identifies regulators of immunomodulatory drug sensitivity

Leukemia volume 33, pages 171–180 (2019)Cite this article

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Abstract

Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4CRBN ubiquitin ligase to trigger proteasomal degradation of the essential transcription factors IKZF1 and IKZF3 and multiple myeloma (MM) cytotoxicity. We have shown that CRBN is also targeted for degradation by SCFFbxo7 ubiquitin ligase. In the current study, we explored the mechanisms underlying sensitivity of MM cells to IMiDs using genome-wide CRISPR-Cas9 screening. We validate that CSN9 signalosome complex, a deactivator of Cullin-RING ubiquitin ligase, inhibits SCFFbxo7 E3 ligase-mediated CRBN degradation, thereby conferring sensitivity to IMiDs; conversely, loss of function of CSN9 signalosome activates SCFFbxo7 complex, thereby enhancing degradation of CRBN and conferring IMiD resistance. Finally, we show that pretreatment with either proteasome inhibitors or NEDD8 activating enzyme (NAE) inhibitors can abrogate degradation and maintain levels of CRBN, thereby enhancing sensitivity to IMiDs. These studies therefore demonstrate that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression.

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References

  1. Palumbo A, Anderson K. Multiple myeloma. N Engl J Med. 2011;364:1046–60.

    Article  CAS  Google Scholar 

  2. Kuehl WM, Bergsagel PL. Multiple myeloma: evolving genetic events and host interactions. Nat Rev Cancer. 2002;2:175–87.

    Article  CAS  Google Scholar 

  3. Fermand JP, Ravaud P, Chevret S, Divine M, Leblond V, Belanger C, et al. High-dose therapy and autologous peripheral blood stem cell transplantation in multiple myeloma: up-front or rescue treatment? Results of a multicenter sequential randomized clinical trial. Blood. 1998;92:3131–6.

    CAS  PubMed  Google Scholar 

  4. Lenhoff S, Hjorth M, Holmberg E, Turesson I, Westin J, Nielsen JL, et al. Impact on survival of high-dose therapy with autologous stem cell support in patients younger than 60 years with newly diagnosed multiple myeloma: a population-based study. Nordic Myeloma Study Group. Blood. 2000;95:7–11.

    CAS  PubMed  Google Scholar 

  5. Fermand JP, Levy Y, Gerota J, Benbunan M, Cosset JM, Castaigne S, et al. Treatment of aggressive multiple myeloma by high-dose chemotherapy and total body irradiation followed by blood stem cells autologous graft. Blood. 1989;73:20–23.

    CAS  PubMed  Google Scholar 

  6. Singhal S, Mehta J, Desikan R, Ayers D, Roberson P, Eddlemon P, et al. Antitumor activity of thalidomide in refractory multiple myeloma. N Engl J Med. 1999;341:1565–71.

    Article  CAS  Google Scholar 

  7. McCarthy PL, Owzar K, Hofmeister CC, Hurd DD, Hassoun H, Richardson PG, et al. Lenalidomide after stem-cell transplantation for multiple myeloma. N Engl J Med. 2012;366:1770–81.

    Article  CAS  Google Scholar 

  8. Lacy MQ, Hayman SR, Gertz MA, Dispenzieri A, Buadi F, Kumar S, et al. Pomalidomide (CC4047) plus low-dose dexamethasone as therapy for relapsed multiple myeloma. J Clin Oncol. 2009;27:5008–14.

    Article  CAS  Google Scholar 

  9. Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, Facon T, et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med. 2005;352:2487–98.

    Article  CAS  Google Scholar 

  10. Herndon TM, Deisseroth A, Kaminskas E, Kane RC, Koti KM, Rothmann MD, et al. U.S. Food and Drug Administration approval: carfilzomib for the treatment of multiple myeloma. Clin Cancer Res. 2013;19:4559–63.

    Article  CAS  Google Scholar 

  11. Shirley M. Ixazomib: First Global Approval. Drugs. 2016;76:405–11.

    Article  CAS  Google Scholar 

  12. Gandolfi S, Laubach JP, Hideshima T, Chauhan D, Anderson KC, Richardson PG. The proteasome and proteasome inhibitors in multiple myeloma. Cancer Metastas- Rev. 2017;36:561–84.

    Article  CAS  Google Scholar 

  13. McKeage K. Daratumumab: first global approval. Drugs. 2016;76:275–81.

    Article  CAS  Google Scholar 

  14. Lokhorst HM, Plesner T, Laubach JP, Nahi H, Gimsing P, Hansson M, et al. Targeting CD38 with daratumumab monotherapy in multiple myeloma. N Engl J Med. 2015;373:1207–19.

    Article  CAS  Google Scholar 

  15. Lonial S, Dimopoulos M, Palumbo A, White D, Grosicki S, Spicka I, et al. Elotuzumab therapy for relapsed or refractory multiple myeloma. N Engl J Med. 2015;373:621–31.

    Article  CAS  Google Scholar 

  16. Laubach JP, Moreau P, San-Miguel JF, Richardson PG. Panobinostat for the treatment of multiple myeloma. Clin Cancer Res. 2015;21:4767–73.

    Article  CAS  Google Scholar 

  17. Attal M, Lauwers-Cances V, Hulin C, Leleu X, Caillot D, Escoffre M, et al. Lenalidomide, bortezomib, and dexamethasone with transplantation for myeloma. N Engl J Med. 2017;376:1311–20.

    Article  CAS  Google Scholar 

  18. Ito T, Ando H, Suzuki T, Ogura T, Hotta K, Imamura Y, et al. Identification of a primary target of thalidomide teratogenicity. Science. 2010;327:1345–50.

    Article  CAS  Google Scholar 

  19. Liu J, Ye J, Zou X, Xu Z, Feng Y, Chen Z, et al. CRL4A(CRBN) E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis. Nat Commun. 2014;5:3924.

    Article  CAS  Google Scholar 

  20. Chen YA, Peng YJ, Hu MC, Huang JJ, Chien YC, Wu JT, et al. The Cullin 4A/B-DDB1-cereblon E3 ubiquitin ligase complex mediates the degradation of CLC-1 chloride channels. Sci Rep. 2015;5:10667.

    Article  Google Scholar 

  21. Higgins JJ, Pucilowska J, Lombardi RQ, Rooney JP. A mutation in a novel ATP-dependent Lon protease gene in a kindred with mild mental retardation. Neurology. 2004;63:1927–31.

    Article  CAS  Google Scholar 

  22. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer. 2007;7:585–98.

    Article  CAS  Google Scholar 

  23. Hideshima T, Chauhan D, Podar K, Schlossman RL, Richardson P, Anderson KC. Novel therapies targeting the myeloma cell and its bone marrow microenvironment. Semin Oncol. 2001;28:607–12.

    Article  CAS  Google Scholar 

  24. Kronke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 2014;343:301–5.

    Article  Google Scholar 

  25. Lu G, Middleton RE, Sun H, Naniong M, Ott CJ, Mitsiades CS, et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science. 2014;343:305–9.

    Article  CAS  Google Scholar 

  26. Kronke J, Fink EC, Hollenbach PW, MacBeth KJ, Hurst SN, Udeshi ND, et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature. 2015;523:183–8.

    Article  CAS  Google Scholar 

  27. Fischer ES, Bohm K, Lydeard JR, Yang H, Stadler MB, Cavadini S, et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature. 2014;512:49–53.

    Article  CAS  Google Scholar 

  28. Chamberlain PP, Lopez-Girona A, Miller K, Carmel G, Pagarigan B, Chie-Leon B, et al. Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat Struct Mol Biol. 2014;21:803–9.

    Article  CAS  Google Scholar 

  29. Petzold G, Fischer ES, Thoma NH. Structural basis of lenalidomide-induced CK1alpha degradation by the CRL4(CRBN) ubiquitin ligase. Nature. 2016;532:127–30.

    Article  CAS  Google Scholar 

  30. Song T, Liang S, Liu J, Zhang T, Yin Y, Geng C, et al. CRL4 antagonizes SCF(Fbxo7)-mediated turnover of cereblon and BK channel to regulate learning and memory. PLoS Genet. 2017;14:e1007165.

    Article  Google Scholar 

  31. Hideshima T, Cottini F, Nozawa Y, Seo HS, Ohguchi H, Samur MK, et al. p53-related protein kinase confers poor prognosis and represents a novel therapeutic target in multiple myeloma. Blood. 2017;129:1308–19.

    Article  CAS  Google Scholar 

  32. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343:84–87.

    Article  CAS  Google Scholar 

  33. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11:783–4.

    Article  CAS  Google Scholar 

  34. Zhu YX, Braggio E, Shi CX, Bruins LA, Schmidt JE, Van Wier S, et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood. 2011;118:4771–9.

    Article  CAS  Google Scholar 

  35. Lopez-Girona A, Mendy D, Ito T, Miller K, Gandhi AK, Kang J, et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia. 2012;26:2326–35.

    Article  CAS  Google Scholar 

  36. Lingaraju GM, Bunker RD, Cavadini S, Hess D, Hassiepen U, Renatus M, et al. Crystal structure of the human COP9 signalosome. Nature. 2014;512:161–5.

    Article  CAS  Google Scholar 

  37. Dubiel D, Rockel B, Naumann M, Dubiel W. Diversity of COP9 signalosome structures and functional consequences. FEBS Lett. 2015;589(19 Pt A):2507–13.

    Article  CAS  Google Scholar 

  38. Cope GA, Deshaies RJ. COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell. 2003;114:663–71.

    Article  CAS  Google Scholar 

  39. Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou C, et al. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science. 2001;292:1382–5.

    Article  CAS  Google Scholar 

  40. Olma MH, Roy M, Le Bihan T, Sumara I, Maerki S, Larsen B, et al. An interaction network of the mammalian COP9 signalosome identifies Dda1 as a core subunit of multiple Cul4-based E3 ligases. J Cell Sci. 2009;122(Pt 7):1035–44.

    Article  CAS  Google Scholar 

  41. Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009;458:732–6.

    Article  CAS  Google Scholar 

  42. Richardson PG, Xie W, Jagannath S, Jakubowiak A, Lonial S, Raje NS, et al. A phase 2 trial of lenalidomide, bortezomib, and dexamethasone in patients with relapsed and relapsed/refractory myeloma. Blood. 2014;123:1461–9.

    Article  CAS  Google Scholar 

  43. Avigan D, Rosenblatt J. Current treatment for multiple myeloma. N Engl J Med. 2014;371:961–2.

    Article  CAS  Google Scholar 

  44. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.

    Article  CAS  Google Scholar 

  45. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.

    Article  CAS  Google Scholar 

  46. Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014;343:80–84.

    Article  CAS  Google Scholar 

  47. Lee MH, Zhao R, Phan L, Yeung SC. Roles of COP9 signalosome in cancer. Cell Cycle. 2011;10:3057–66.

    Article  CAS  Google Scholar 

  48. Lee EK, Diehl JA. SCFs in the new millennium. Oncogene. 2014;33:2011–8.

    Article  CAS  Google Scholar 

  49. Skaar JR, Pagan JK, Pagano M. Mechanisms and function of substrate recruitment by F-box proteins. Nat Rev Mol Cell Biol. 2013;14:369–81.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank X.H. Feng and Z.P. Xia for expression vectors. We thank the staff from the hematologic neoplasia core facilities at Dana-Farber Cancer Institute for technical assistance. We also thank the Genome Center of WuXi AppTec Inc. for the initial data analysis of the CRISPR screening.

Funding

This study was supported by the National Institute of Health Grant; SPORE-P50100707 (KCA), P01-CA078378 (K.C.A.), R01-CA050947 (K.C.A), and R01-CA178264 (T.H. and K.C.A.). K.C.A. is an American Cancer Society Clinical Research Professor. This study was also supported in part by funds from the National 973 Plan for Basic Research (2015CB553803), National Natural Science Foundation of China (31671334), Fundamental Research Funds for the Central Universities, and Key Construction Program of the National ‘985’ Project.

Author contributons

J.L., T.S., and W.Z. designed and performed the research, analyzed the data, and wrote the manuscript; L.X. and M.H. performed some experiment; S.W. performed gene expression correlation analysis; Z.P. designed research and analyzed data; Y.T. provided biological material and analyzed data; T.H. designed the research, analyzed the data, wrote the manuscript, and supervised the project; K.C.A and Y.C. conceived the project, designed the research, analyzed the data, wrote the manuscript, and supervised the project.

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Author notes

  1. These authors contributed equally: Jiye Liu, Tianyu Song, Wenrong Zhou, Kenneth C. Anderson, Yong Cang.

Authors and Affiliations

  1. Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Jiye Liu, Lijie Xing, Matthew Ho, Yu-Tzu Tai, Teru Hideshima & Kenneth C. Anderson

  2. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Tianyu Song & Yong Cang

  3. Oncology Business Unit and Innovation Center for Cell Signalling Network, WuXi AppTec Group, Shanghai, China

    Wenrong Zhou & Zhengang Peng

  4. Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China

    Lijie Xing

  5. Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA

    Su Wang

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K.C.A. serves on advisory boards to Celgene and Millennium. All other authors declare no competing financial interests.

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Liu, J., Song, T., Zhou, W. et al. A genome-scale CRISPR-Cas9 screening in myeloma cells identifies regulators of immunomodulatory drug sensitivity. Leukemia 33, 171–180 (2019). https://doi.org/10.1038/s41375-018-0205-y

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