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.

SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate

Abstract

Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly ‘undruggable’ C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Human and rabbit SALL4 zinc fingers are direct, thalidomide-dependent substrates of cereblon-CRL4 in vitro, whereas mouse SALL4 is not.
Fig. 2: SALL4 is degraded in cell lines in a cereblon- and proteasome-dependent fashion upon treatment with thalidomide.
Fig. 3: SALL4 and ZFP91 examined by immunohistochemistry in rabbit, wild-type mouse, and humanized cereblon mouse testes.
Fig. 4: SALL4 localizes to the limb bud during development in rabbit embryos, and decreases upon treatment with thalidomide.
Fig. 5: Model for thalidomide-induced SALL4 ubiquitination by cereblon-CRL4 and subsequent cellular degradation by the 26 S proteasome.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

References

  1. 1.

    Lenz, W. A short history of thalidomide embryopathy. Teratology 38, 203–215 (1988).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  2. 2.

    Vargesson, N. Thalidomide-induced teratogenesis: history and mechanisms. Birth Defects Res. C Embryo Today 105, 140–156 (2015).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  3. 3.

    Lenz, W. P., Pfeiffer, R. A., Kosenow, W. & Hayman, D. J. Thalidomide and congenital abnormalities. Lancet 279, 45–46 (1962).

    Article  Google Scholar 

  4. 4.

    Mcbride, W. G. Thalidomide and congenital abnormalities. Lancet 278, 1358 (1961).

    Article  Google Scholar 

  5. 5.

    Kim, J. H. & Scialli, A. R. Thalidomide: the tragedy of birth defects and the effective treatment of disease. Toxicol. Sci. 122, 1–6 (2011).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  6. 6.

    Miller, M. T. & Strömland, K. Teratogen update: thalidomide: a review, with a focus on ocular findings and new potential uses. Teratology 60, 306–321 (1999).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  7. 7.

    Singhal, S. et al. Antitumor activity of thalidomide in refractory multiple myeloma. N. Engl. J. Med. 341, 1565–1571 (1999).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  8. 8.

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

    CAS  Article  Google Scholar 

  9. 9.

    Angers, S. et al. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443, 590–593 (2006).

    CAS  Google Scholar 

  10. 10.

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

    CAS  Article  PubMed Central  Google Scholar 

  11. 11.

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

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  12. 12.

    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).

    CAS  Article  PubMed Central  Google Scholar 

  13. 13.

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

    CAS  Article  PubMed Central  Google Scholar 

  14. 14.

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

    Article  PubMed Central  CAS  Google Scholar 

  15. 15.

    Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523, 183–188 (2015).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. 16.

    Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535, 252–257 (2016).

    CAS  Article  PubMed Central  Google Scholar 

  17. 17.

    An, J. et al. pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4CRBN ubiquitin ligase. Nat. Commun. 8, 15398 (2017).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  18. 18.

    Petzold, G., Fischer, E. S., & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532, 127–130 (2016).

    CAS  Article  Google Scholar 

  19. 19.

    Tan, X. et al. Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446, 640–645 (2007).

    CAS  Article  Google Scholar 

  20. 20.

    Winter, G. E. et al. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  21. 21.

    Lu, J. et al. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 22, 755–763 (2015).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  22. 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).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  23. 23.

    Sterz, H., Nothdurft, H., Lexa, P. & Ockenfels, H. Teratologic studies on the Himalayan rabbit: new aspects of thalidomide-induced teratogenesis. Arch. Toxicol. 60, 376–381 (1987).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  24. 24.

    Akiyama, R. et al. Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements. Proc. Natl Acad. Sci. USA 112, 5075–5080 (2015).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  25. 25.

    Kohlhase, J. et al. Okihiro syndrome is caused by SALL4 mutations. Hum. Mol. Genet. 11, 2979–2987 (2002).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  26. 26.

    Koshiba-Takeuchi, K. et al. Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart. Nat. Genet. 38, 175–183 (2006).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  27. 27.

    Elling, U., Klasen, C., Eisenberger, T., Anlag, K. & Treier, M. Murine inner cell mass-derived lineages depend on Sall4 function. Proc. Natl Acad. Sci. USA 103, 16319–16324 (2006).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  28. 28.

    Kohlhase, J. et al. SALL4 mutations in Okihiro syndrome (Duane-radial ray syndrome), acro-renal-ocular syndrome, and related disorders. Hum. Mutat. 26, 176–183 (2005).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  29. 29.

    Kohlhase, J. et al. Mutations at the SALL4 locus on chromosome 20 result in a range of clinically overlapping phenotypes, including Okihiro syndrome, Holt-Oram syndrome, acro-renal-ocular syndrome, and patients previously reported to represent thalidomide embryopathy. J. Med. Genet. 40, 473–478 (2003).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  30. 30.

    Kohlhase, J. & Holmes, L. B. Mutations in SALL4 in malformed father and daughter postulated previously due to reflect mutagenesis by thalidomide. Birth Defects Res. A Clin. Mol. Teratol. 70, 550–551 (2004).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. 31.

    Borozdin, W. et al. Novel mutations in the gene SALL4 provide further evidence for acro-renal-ocular and Okihiro syndromes being allelic entities, and extend the phenotypic spectrum. J. Med. Genet. 41, e102 (2004).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  32. 32.

    Borozdin, W. et al. SALL4 deletions are a common cause of Okihiro and acro-renal-ocular syndromes and confirm haploinsufficiency as the pathogenic mechanism. J. Med. Genet. 41, e113 (2004).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  33. 33.

    Paradisi, I. & Arias, S. IVIC syndrome is caused by a c.2607delA mutation in the SALL4 locus. Am. J. Med. Genet. A. 143, 326–332 (2007).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. 34.

    Al-Baradie, R. et al. Duane radial ray syndrome (Okihiro syndrome) maps to 20q13 and results from mutations in SALL4, a new member of the SAL family. Am. J. Hum. Genet. 71, 1195–1199 (2002).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  35. 35.

    Akuffo, A. A. et al. Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon. J. Biol. Chem. 293, 6187–6200 (2018).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  36. 36.

    Schumacher, H., Smith, R. L. & Williams, R. T. The metabolism of thalidomide: the spontaneous hydrolysis of thalidomide in solution. Br. J. Pharmacol. Chemother. 25, 324–337 (1965).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  37. 37.

    Arlen, R. R. & Wells, P. G. Inhibition of thalidomide teratogenicity by acetylsalicylic acid: evidence for prostaglandin H synthase-catalyzed bioactivation of thalidomide to a teratogenic reactive intermediate. J. Pharmacol. Exp. Ther. 277, 1649–1658 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Lee, C. J., Gonçalves, L. L. & Wells, P. G. Embryopathic effects of thalidomide and its hydrolysis products in rabbit embryo culture: evidence for a prostaglandin H synthase (PHS)-dependent, reactive oxygen species (ROS)-mediated mechanism. FASEB. J. 25, 2468–2483 (2011).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  39. 39.

    Parman, T., Wiley, M. J. & Wells, P. G. Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nat. Med. 5, 582–585 (1999).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  40. 40.

    Matyskiela, M. E. et al. A cereblon modulator (CC-220) with improved degradation of Ikaros and Aiolos. J. Med. Chem. 61, 535–542 (2018).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  41. 41.

    Gassei, K. & Orwig, K. E. SALL4 expression in gonocytes and spermatogonial clones of postnatal mouse testes. PLoS One 8, e53976 (2013).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  42. 42.

    Eildermann, K. et al. Developmental expression of the pluripotency factor sal-like protein 4 in the monkey, human and mouse testis: restriction to premeiotic germ cells. Cells Tissues Organs 196, 206–220 (2012).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  43. 43.

    Higgins, J. J., Pucilowska, J., Lombardi, R. Q. & Rooney, J. P. A mutation in a novel ATP-dependent Lon protease gene in a kindred with mild mental retardation. Neurology 63, 1927–1931 (2004).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  44. 44.

    Sheereen, A. et al. A missense mutation in the CRBN gene that segregates with intellectual disability and self-mutilating behaviour in a consanguineous Saudi family. J. Med. Genet. 54, 236–240 (2017).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  45. 45.

    Ito, T., Ando, H. & Handa, H. Teratogenic effects of thalidomide: molecular mechanisms. Cell. Mol. Life Sci. 68, 1569–1579 (2011).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  46. 46.

    Gandhi, A. K. et al. Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity. Br. J. Haematol. 164, 233–244 (2014).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  47. 47.

    Wilson, J. G. Embryological considerations in teratology. Ann. NY Acad. Sci. 123, 219–227 (1965).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  48. 48.

    Staples, R. E. & Schnell, V. L. Refinements in rapid clearing technic in the Koh-Alizarin Red S method for fetal bone. Stain Technol. 39, 61–63 (1964).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Many thanks to K. Kolaja, N. Collins and the teratogenicity working group for discussions relating to this work.

Author information

Affiliations

Authors

Contributions

M.E.M., A.C., T.C., W.F., P.A., and M. Riley. performed biochemical studies. X.Z., G.L., and C.-C.L. performed cellular experiments. S.C., C.D., Y.R., M.W., and C.-W.L. performed IHC studies. J. Hui., K.S., and K.B. planned in vivo experiments. M.E.M., S.C., G.L., J. Hui, J. Hartke, G.K., M. Rolfe., R.V., L.G.H., and P.P.C. planned studies, and all authors analyzed the data and prepared the manuscript.

Corresponding author

Correspondence to Philip P. Chamberlain.

Ethics declarations

Competing interests

Authors are or have been employees or consultants of Celgene.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Tables 1–2

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Matyskiela, M.E., Couto, S., Zheng, X. et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat Chem Biol 14, 981–987 (2018). https://doi.org/10.1038/s41589-018-0129-x

Download citation

Further reading

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