Abstract

Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4CRBN. These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350 (2010)

  2. 2.

    et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014)

  3. 3.

    et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014)

  4. 4.

    et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4CRBN. Br. J. Haematol. 164, 811–821 (2014)

  5. 5.

    et al. Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N. Engl. J. Med. 355, 1456–1465 (2006)

  6. 6.

    et al. Efficacy of lenalidomide in myelodysplastic syndromes. N. Engl. J. Med. 352, 549–557 (2005)

  7. 7.

    et al. A randomized phase 3 study of lenalidomide versus placebo in RBC transfusion-dependent patients with low-/intermediate-1-risk myelodysplastic syndromes with del5q. Blood 118, 3765–3776 (2011)

  8. 8.

    et al. Lenalidomide inhibits the malignant clone and up-regulates the SPARC gene mapping to the commonly deleted region in 5q- syndrome patients. Proc. Natl Acad. Sci. USA 104, 11406–11411 (2007)

  9. 9.

    et al. A critical role for phosphatase haplodeficiency in the selective suppression of deletion 5q MDS by lenalidomide. Proc. Natl Acad. Sci. USA 106, 12974–12979 (2009)

  10. 10.

    et al. Narrowing and genomic annotation of the commonly deleted region of the 5q- syndrome. Blood 99, 4638–4641 (2002)

  11. 11.

    et al. Integrated genomic analysis implicates haploinsufficiency of multiple chromosome 5q31.2 genes in de novo myelodysplastic syndromes pathogenesis. PLoS ONE 4, e4583 (2008)

  12. 12.

    et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002)

  13. 13.

    , , & Large-scale identification of ubiquitination sites by mass spectrometry. Nature Protocols 8, 1950–1960 (2013)

  14. 14.

    et al. Gene expression profiling of CD34+ cells in patients with the 5q− syndrome. Br. J. Haematol. 139, 578–589 (2007)

  15. 15.

    et al. Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS. Cancer Cell 26, 509–520 (2014)

  16. 16.

    et al. Csnk1a1 inhibition has p53-dependent therapeutic efficacy in acute myeloid leukemia. J. Exp. Med. 211, 605–612 (2014)

  17. 17.

    , , & CK1α plays a central role in mediating MDM2 control of p53 and E2F–1 protein stability. J. Biol. Chem. 284, 32384–32394 (2009)

  18. 18.

    , , , & Casein kinase 1α regulates an MDMX intramolecular interaction to stimulate p53 binding. Mol. Cell. Biol. 32, 4821–4832 (2012)

  19. 19.

    et al. CKIα ablation highlights a critical role for p53 in invasiveness control. Nature 470, 409–413 (2011)

  20. 20.

    et al. The CK1 family: contribution to cellular stress response and its role in carcinogenesis. Front. Oncol. 4, 96 (2014)

  21. 21.

    , & Casein kinase: the triple meaning of a misnomer. Biochem. J. 460, 141–156 (2014)

  22. 22.

    , & Teratogenic effects of thalidomide in rabbits, rats, hamsters, and mice. Toxicol. Appl. Pharmacol. 7, 268–286 (1965)

  23. 23.

    et al. Drug response in a genetically engineered mouse model of multiple myeloma is predictive of clinical efficacy. Blood 120, 376–385 (2012)

  24. 24.

    et al. Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014)

  25. 25.

    et al. Structure of the human Cereblon–DDB1–lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nature Struct. Mol. Biol 21, 803–809 (2014)

  26. 26.

    et al. TP53 mutations in low-risk myelodysplastic syndromes with del(5q) predict disease progression. J. Clin. Oncol. 29, 1971–1979 (2011)

  27. 27.

    & Isobaric labeling-based relative quantification in shotgun proteomics. J. Proteome Res. 13, 5293–5309 (2014)

  28. 28.

    Gene deletion: a new target for cancer chemotherapy. Lancet 342, 662–664 (1993)

  29. 29.

    et al. Cancer vulnerabilities unveiled by genomic loss. Cell 150, 842–854 (2012)

  30. 30.

    et al. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 451, 335–339 (2008)

  31. 31.

    & Ribosomopathies: human disorders of ribosome dysfunction. Blood 115, 3196–3205 (2010)

  32. 32.

    et al. Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma. Cancer Cell 21, 723–737 (2012)

  33. 33.

    et al. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nature Methods 10, 634–637 (2013)

  34. 34.

    & Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 307–310 (1986)

  35. 35.

    Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology 3, Article 3 (2004)

  36. 36.

    & Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc., B (1995)

  37. 37.

    et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nature Biotechnol. (2014)

  38. 38.

    et al. Quantitative temporal viromics: an approach to investigate host-pathogen interaction. Cell 157, 1460–1472 (2014)

Download references

Acknowledgements

We thank D. Heckl for cloning of the EGI vector and technical advice, R. Mathieu and M. Paktinat for help with FACS sorting, M. Chen for help with colony management and animal care, S. Köpff for technical support, and C. Fontanillo for proteomic analysis computational support. Patient samples were provided by the Stem Cell and Xenograft Core of the University of Pennsylvania. This work was supported by the NIH (R01HL082945 and P01CA108631), the Edward P. Evans Foundation, the Gabrielle’s Angel Foundation, and a Leukemia and Lymphoma Society Scholar Award to B.L.E.; J.K. was supported by the German Research Foundation (DFG, Emmy Noether Fellowship Kr3886/2-1, Kr3886/1-1, and SFB1074) and the Else-Kröner Fresenius Foundation. E.C.F. was supported by award T32GM007753 from the National Institute of General Medical Sciences.

Author information

Author notes

    • Jan Krönke
    •  & Emma C. Fink

    These authors contributed equally to this work.

Affiliations

  1. Brigham and Women’s Hospital, Division of Hematology, Boston, Massachusetts 02115, USA

    • Jan Krönke
    • , Emma C. Fink
    • , Slater N. Hurst
    • , Rebekka K. Schneider
    • , Marie McConkey
    • , Marcus Järås
    •  & Benjamin L. Ebert
  2. University Hospital of Ulm, Department of Internal Medicine III, 89081 Ulm, Germany

    • Jan Krönke
    •  & Lars Bullinger
  3. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Jan Krönke
    • , Emma C. Fink
    • , Namrata D. Udeshi
    • , D. R. Mani
    • , Tanya Svinkina
    • , Steven A. Carr
    •  & Benjamin L. Ebert
  4. Celgene Corporation, San Diego, California 92121, USA

    • Paul W. Hollenbach
    • , Kyle J. MacBeth
    • , Philip P. Chamberlain
    • , Hon Wah Man
    • , Anita K. Gandhi
    • , Brian E. Cathers
    •  & Rajesh Chopra
  5. Roswell Park Cancer Institute, Buffalo, New York 14263, USA

    • Elizabeth Griffiths
    •  & Meir Wetzler

Authors

  1. Search for Jan Krönke in:

  2. Search for Emma C. Fink in:

  3. Search for Paul W. Hollenbach in:

  4. Search for Kyle J. MacBeth in:

  5. Search for Slater N. Hurst in:

  6. Search for Namrata D. Udeshi in:

  7. Search for Philip P. Chamberlain in:

  8. Search for D. R. Mani in:

  9. Search for Hon Wah Man in:

  10. Search for Anita K. Gandhi in:

  11. Search for Tanya Svinkina in:

  12. Search for Rebekka K. Schneider in:

  13. Search for Marie McConkey in:

  14. Search for Marcus Järås in:

  15. Search for Elizabeth Griffiths in:

  16. Search for Meir Wetzler in:

  17. Search for Lars Bullinger in:

  18. Search for Brian E. Cathers in:

  19. Search for Steven A. Carr in:

  20. Search for Rajesh Chopra in:

  21. Search for Benjamin L. Ebert in:

Contributions

J.K., E.C.F., and B.L.E. initiated the project. J.K., E.C.F., P.W.H., K.J.M., S.N.H, M.McC., A.K.G., M.J., and R.K.S. designed and performed cell experiments and protein analysis. E.C.F. designed and performed mouse competition and patient sample experiments. N.D.U., D.R.M., and T.S. performed KG-1 proteomics and analysis. E.G. and M.W. provided patient samples. P.P.C. performed the structural analysis. H.W.M. synthesized CC-122. K.J.M. provided MDS-L proteomics. B.E.C., R.C., L.B., S.A.C., and B.L.E. supervised the work. J.K., E.C.F., and B.L.E. wrote the manuscript. All authors assisted in editing the manuscript.

Competing interests

P.W.H., K.M., P.P.C., H.W.M., A.K.G., B.E.C., and R.C. are employed by Celgene Corporation. B.L.E. has consulted for Celgene. J.K. received honoraria from Celgene. All other authors have no competing interests to declare.

Corresponding author

Correspondence to Benjamin L. Ebert.

The original mass spectra may be downloaded from MassIVE (http://massive.ucsd.edu) using the identifier: MSV000079014. The data are accessible at ftp://massive.ucsd.edu/MSV000079014.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary information

    This file contains Supplementary Methods (Synthesis and characterization of CC-1220); a Supplementary Note (Pharmacokinetics of lenalidomide in humansand) and Supplementary Figure 1, which shows the full uncropped scans of all Western Blots with molecular weight markers.

Excel files

  1. 1.

    Supplementary Table 1

    This file contains the full data table for KG-1 proteomics studies. Proteome tab lists the effects of 1 μM and 10 μM lenalidomide on protein levels. KGG tab lists the results of ubiquitin profiling.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature14610

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.