Article | Published:

Multiple myeloma gammopathies

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


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

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

  2. 2.

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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 11.

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

  12. 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.

  13. 13.

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

  14. 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.

  15. 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.

  16. 16.

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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  21. 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.

  22. 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.

  23. 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.

  24. 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.

  25. 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.

  26. 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.

  27. 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.

  28. 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.

  29. 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.

  30. 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.

  31. 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.

  32. 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.

  33. 33.

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

  34. 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.

  35. 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.

  36. 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.

  37. 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.

  38. 38.

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

  39. 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.

  40. 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.

  41. 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.

  42. 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.

  43. 43.

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

  44. 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.

  45. 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.

  46. 46.

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

  47. 47.

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

  48. 48.

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

  49. 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.

Download references


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.


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.

Author information

Correspondence to Kenneth C. Anderson or Yong Cang.

Ethics declarations

Conflict of interest

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

Electronic supplementary material

Supplementary information

Supplementary Table S1

Supplementary Table S2

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5