Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Lenalidomide-induced changes in ubiquitination and protein levels.
Figure 2: Lenalidomide induces the ubiquitination of CK1α by CRL4CRBN.
Figure 3: Ectopic CSNK1A1 overexpression reduces lenalidomide sensitivity in primary MDS del(5q) cells.
Figure 4: Amino acid changes in CRBN explain species-specific lenalidomide effects.
Figure 5: Effects of lenalidomide treatment on Csnk1a1+/− mouse haematopoietic cells.
Figure 6: Substrate specificity of thalidomide analogues.

References

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

    Article  CAS  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  4. Gandhi, A. K. 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)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Fenaux, P. 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)

    Article  CAS  Google Scholar 

  8. Pellagatti, A. 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)

    Article  CAS  ADS  Google Scholar 

  9. Wei, S. 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)

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  Google Scholar 

  12. Ong, S. E. 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)

    Article  CAS  Google Scholar 

  13. Udeshi, N. D., Mertins, P., Svinkina, T. & Carr, S. A. Large-scale identification of ubiquitination sites by mass spectrometry. Nature Protocols 8, 1950–1960 (2013)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  17. Huart, A. S., MacLaine, N. J., Meek, D. W. & Hupp, T. R. CK1α plays a central role in mediating MDM2 control of p53 and E2F–1 protein stability. J. Biol. Chem. 284, 32384–32394 (2009)

    Article  CAS  Google Scholar 

  18. Wu, S., Chen, L., Becker, A., Schonbrunn, E. & Chen, J. Casein kinase 1α regulates an MDMX intramolecular interaction to stimulate p53 binding. Mol. Cell. Biol. 32, 4821–4832 (2012)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

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

    Article  Google Scholar 

  21. Venerando, A., Ruzzene, M. & Pinna, L. A. Casein kinase: the triple meaning of a misnomer. Biochem. J. 460, 141–156 (2014)

    Article  CAS  Google Scholar 

  22. Fratta, I. D., Sigg, E. B. & Maiorana, K. Teratogenic effects of thalidomide in rabbits, rats, hamsters, and mice. Toxicol. Appl. Pharmacol. 7, 268–286 (1965)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  25. Chamberlain, P. P. 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)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  27. Rauniyar, N. & Yates, J. R., III. Isobaric labeling-based relative quantification in shotgun proteomics. J. Proteome Res. 13, 5293–5309 (2014)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  31. Narla, A. & Ebert, B. L. Ribosomopathies: human disorders of ribosome dysfunction. Blood 115, 3196–3205 (2010)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Bland, J. M. & Altman, D. G. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 307–310 (1986)

    Article  CAS  Google Scholar 

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

    Article  MathSciNet  Google Scholar 

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

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

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

    Article  CAS  Google Scholar 

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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Benjamin L. Ebert.

Ethics declarations

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.

Additional information

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 figures and tables

Extended Data Figure 1 Effect of lenalidomide on specific ubiquitination sites.

Median log2 ratios for different lysine residues in CK1α isoform 2, IKZF1 isoform 1, and CRBN isoform 2 for 1 or 10 µM lenalidomide-treated KG-1 cells versus DMSO-treated cells. SILAC experiments were performed in two biological replicates with flipped SILAC labelling. Only lysine residues detected in both replicates are shown. Error bars show range.

Extended Data Figure 2 Effect of lenalidomide in human cells.

a, Time course of effect of lenalidomide treatment on CK1α protein levels in KG-1 cells. b, Immunoblot of CK1α protein levels in the bone marrow (1, 2) and peripheral blood (3, 4) mononuclear cells of AML patients treated with lenalidomide as part of a clinical trial. Pre-treatment samples are taken at the screen or before the first treatment (C1D1). Subsequent time points are cycle 1 day 15 (C1D15), cycle 2 day 1 (C2D1) or cycle 1 day 8 (C1D8) of lenalidomide treatment. Further details about these patients (n = 4) can be found in Extended Data Table 2. c, MM1S, K562, and Jurkat cells were treated with different concentrations of lenalidomide for 24 h. CK1α protein levels were detected by western blot and CSNK1A1 mRNA expression levels were measured by RQ-PCR. Data are mean ± s.d., n = 3 each with three technical replicates. d, Immunoblot confirming loss of CRBN expression in 293T cells with the CRBN gene disrupted by CRIPSR-Cas9 genome editing. e, Immunoprecipitation with a CRBN-specific antibody in 293T cells treated with DMSO or 10 µM lenalidomide for 5 h in the presence of 10 µM MG132. Results in a, c, d, and e are each representative of two independent experiments. Uncropped blots are shown in Supplementary Fig. 1.

Extended Data Figure 3 Sequence determinants of CK1α degradation.

a, 293T cells were transfected with plasmids expressing Flag–CK1α isoform 1 or isoform 2 together with a human CRBN-expressing plasmid. Cells were treated with DMSO or 10 µM lenalidomide for 16 h. Cells expressing Flag–CK1α isoform 1, which contains a nuclear localization domain, were incubated in the absence or presence of the nuclear export inhibitor leptomycin B. b, 293T cells expressing Flag–CK1α isoform 2 wild-type or two different point mutations identified in patient samples were treated with DMSO or 10 µM lenalidomide for 16 h. c, Immunofluorescence for CK1α after treatment with DMSO or 10 μM lenalidomide. Enlarged area is indicated by a box in Merge. FITC channel represents staining for CK1α. No changes in CK1α localization are seen upon lenalidomide treatment. Experiment was performed twice in biological duplicate. In each condition, at least 25 cells were assessed. d, Chimaeric proteins of casein kinase 1A1 (CK1α) and casein kinase 1E (CK1ε), which shares significant homology with CK1α but is not responsive to lenalidomide, that were used in e to determine the lenalidomide-responsive region in CK1α. e, Flag-tagged (chimaeric) proteins from d were transfected in 293T cells together with a CRBN-expressing plasmid. Cells were treated with 1 µM lenalidomide for 24 h and protein was detected with a Flag-specific antibody. Data are representative of two (a, c), three (b) or four (e) independent experiments. Uncropped blots are shown in Supplementary Fig. 1.

Extended Data Figure 4 CSNK1A1 knockdown increases lenalidomide sensitivity in haematopoietic cells.

a, Knockdown validation by western blot. bd, CD34+ cells were transduced with GFP-labelled lentivirus expressing either control shRNA targeting luciferase (b) or shRNA targeting CSNK1A1 (c, d) and treated with DMSO or 1 µM lenalidomide. The percentage of GFP+ cells was assessed by flow cytometry over time. Results are representative of 3 independent experiments each with n = 3 biological replicates.

Extended Data Figure 5 Expression of CSNK1A1 and IKZF1 in patient samples.

a, mRNA expression of CSNK1A1 in cord blood CD34+ cells infected with lentivirus expressing human CSNK1A1 or empty vector. CD34+ cells were infected with GFP-tagged lentivirus and GFP+ cells were sorted three days later. Values are mean ± s.d., n = 4 biological replicates, each with 3 technical replicates. b, mRNA expression of IKZF1 in cord blood CD34+ cells infected with lentivirus expressing IKZF1 or empty vector as in a. Values are mean ± s.d., n = 3 biological replicates, each with 3 technical replicates. c, CD34+ cells derived from patient or control bone marrow were transduced with a lentivirus expressing human IKZF1 (hIKZF1) and GFP or an empty control vector and treated with DMSO or 1 µM lenalidomide. The percentage of GFP+ cells was assessed by flow cytometry after five days for each vector-drug combination. Results are reported as a ratio of the percentage of GFP+ cells in the lenalidomide condition to the percentage of GFP+ cells in the DMSO condition. Results are combined from three experiments. d, Characteristics of patient samples used for CSNK1A1 and IKZF1 expression experiments. Results of TP53 sequencing, including exons with adequate coverage, is given in the rightmost column. All samples sequenced had wild-type TP53. ND, not done due to limited patient material. WT, TP53 exon sequence has only known benign polymorphisms.

Extended Data Figure 6 Effect of lenalidomide on mouse cells.

a, CK1α protein levels are unaffected in mouse Ba/F3 cells and primary mouse AML cells (MA9) treated with a range of lenalidomide doses. Data are representative of two independent experiments (n = 2). b, CK1α expression in bone marrow cells of mice treated with DMSO (n = 5) or lenalidomide (n = 5). c, CK1α protein levels in Ba/F3 cells transduced with empty vector, mouse Crbn, human CRBN or Crbn(I391V) and treated with lenalidomide. d, Quantification of CK1α protein levels in Ba/F3 cells using ImageJ. Graphs show the fraction of normalized CK1α protein levels as compared to control (DMSO) treated cells of the respective line. Bars represent mean ± s.e.m. from three independent experiments as in c. e, f, Effect of lenalidomide on an IKZF3–luciferase (e) and IKZF1–luciferase fusion protein (f) in 293T cells expressing human, mouse or different chimaeras or mutations of CRBN. Data are shown as mean ± s.e.m. (n = 3, biological replicates) and are representative of three (f) or five (e) independent experiments. Uncropped blots are shown in Supplementary Fig. 1.

Extended Data Figure 7 Difference electron density map of mouse residue I391 calculated in the absence of a side chain showing the favoured orientation of the residue.

The density is contoured at 3.8σ following a single round of Refmac5 refinement.

Extended Data Figure 8 Comparison of the effects of thalidomide derivatives.

a, Comparison of log2 ratios for CK1α and IKZF1 in MDS-L cells after treatment with lenalidomide or CC-122 for 24 or 72 h assessed by tandem mass tag (TMT) quantitative proteomics. Analysis was performed with n = 4 for DMSO control and n = 3 for each drug treatment time point. b, Adjusted P values for CK1α and IKZF1 proteomic data in MDS-L cells. c, Western blot validation of IKZF1 and CK1α levels in DMSO (n = 4), lenalidomide (n = 3) and CC-122 (n = 3) treated samples used for MDS-L proteomic analysis. d, Western blot validation of the effects of the different agents on CK1α and IKZF1 protein levels in KG-1 cells. e, Effect of lenalidomide, pomalidomide (Pom), and thalidomide (Thal) on protein levels of CK1α, β-catenin, and IKZF1 in KG-1 cells treated for 24 h with the indicated drug concentrations. f, Effect of CC-122 and lenalidomide on β-catenin protein levels in KG-1 cells after 72 h. g, Effect of lenalidomide on CK1α and β-catenin protein levels in HEL cells. Data are representative of two (e, g) or three (c, d) independent experiments. Uncropped blots are shown in Supplementary Fig. 1.

Extended Data Table 1 Statistically significant SILAC results with 1 μM lenalidomide
Extended Data Table 2 Characteristics of the patient samples from the AML-001 trial used in Extended Data Fig. 2b

Supplementary information

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. (PDF 935 kb)

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. (XLSX 8344 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Krönke, J., Fink, E., Hollenbach, P. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523, 183–188 (2015). https://doi.org/10.1038/nature14610

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature14610

This article is cited by

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing