p63 is a cereblon substrate involved in thalidomide teratogenicity

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Abstract

Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity.

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Fig. 1: ∆Np63α and TAp63α are downstream factors of the thalidomide–CRL4CRBN pathway.
Fig. 2: ∆Np63α and TAp63α are neosubstrates of CRL4CRBN.
Fig. 3: Overexpression of z∆Np63 reverses thalidomide-induced fin malformation in zebrafish.
Fig. 4: z∆Np63, but not zTAp63, suppresses fin malformations in zebrafish.
Fig. 5: Overexpression of zTAp63 reverses thalidomide-induced developmental defects of otic vesicles in zebrafish.
Fig. 6: Model of the molecular mechanism of thalidomide teratogenicity.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information or are available from the corresponding author on reasonable request.

References

  1. 1.

    Rehman, W., Arfons, L. M. & Lazarus, H. M. The rise, fall and subsequent triumph of thalidomide: lessons learned in drug development. Ther. Adv. Hematol. 2, 291–308 (2011).

  2. 2.

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

  3. 3.

    Bartlett, J. B., Dredge, K. & Dalgleish, A. G. The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nat. Rev. Cancer 4, 314–322 (2004).

  4. 4.

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

  5. 5.

    Petroski, M. D. & Deshaies, R. J. Function and regulation of cullin-RING ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 6, 9–20 (2005).

  6. 6.

    Moon, A. M. & Capecchi, M. R. Fgf8 is required for outgrowth and patterning of the limbs. Nat. Genet. 26, 455–459 (2000).

  7. 7.

    Lewandoski, M., Sun, X. & Martin, G. R. Fgf8 signalling from the AER is essential for normal limb development. Nat. Genet. 26, 460–463 (2000).

  8. 8.

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

  9. 9.

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

  10. 10.

    Kronke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature 523, 183–188 (2015).

  11. 11.

    Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase. Nature 535, 252–257 (2016).

  12. 12.

    An, J. et al. pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4(CRBN) ubiquitin ligase. Nat. Commun. 8, 15398 (2017).

  13. 13.

    Donovan, K. A. et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. Elife 7, e38430 (2018).

  14. 14.

    Matyskiela, M. E. et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 14, 981–987 (2018).

  15. 15.

    Rinne, T., Hamel, B., van Bokhoven, H. & Brunner, H. G. Pattern of p63 mutations and their phenotypes—update. Am. J. Med. Genet. A. 140, 1396–1406 (2006).

  16. 16.

    Restelli, M. et al. DLX5, FGF8 and the Pin1 isomerase control ΔNp63α protein stability during limb development: a regulatory loop at the basis of the SHFM and EEC congenital malformations. Hum. Mol. Genet. 23, 3830–3842 (2014).

  17. 17.

    Mills, A. A. et al. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713 (1999).

  18. 18.

    Boughner, J. C. et al. p63 expression plays a role in developmental rate, embryo size, and local morphogenesis. Dev. Dyn. 257, 779–787 (2018).

  19. 19.

    Terrinoni, A. et al. Role of p63 and the Notch pathway in cochlea development and sensorineural deafness. Proc. Natl Acad. Sci. USA 110, 7300–7305 (2013).

  20. 20.

    Rouleau, M. et al. TAp63 is important for cardiac differentiation of embryonic stem cells and heart development. Stem Cells 29, 1672–1683 (2011).

  21. 21.

    Lapi, E. et al. S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation. Oncogene 25, 3628–3637 (2006).

  22. 22.

    Ichikawa, T., Suenaga, Y., Koda, T., Ozaki, T. & Nakagawara, A. TAp63-dependent induction of growth differentiation factor 15 (GDF15) plays a critical role in the regulation of keratinocyte differentiation. Oncogene 27, 409–420 (2008).

  23. 23.

    Nasevicius, A. & Ekker, S. C. Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26, 216–220 (2000).

  24. 24.

    Siamwala, J. H. et al. Nitric oxide rescues thalidomide mediated teratogenicity. Sci. Rep. 2, 679 (2012).

  25. 25.

    Jiang, L. L. et al. Gambogic acid causes fin developmental defect in zebrafish embryo partially via retinoic acid signaling. Reprod. Toxicol. 63, 161–168 (2016).

  26. 26.

    Bakkers, J., Hild, M., Kramer, C., Furutani-Seiki, M. & Hammerschmidt, M. Zebrafish ΔNp63 is a direct target of Bmp signaling and encodes a transcriptional repressor blocking neural specification in the ventral ectoderm. Dev. Cell 2, 617–627 (2002).

  27. 27.

    Ando, H. & Okamoto, H. Efficient transfection strategy for the spatiotemporal control of gene expression in zebrafish. Mar. Biotechnol. (NY) 8, 295–303 (2006).

  28. 28.

    Chung, F. et al. Thalidomide pharmacokinetics and metabolite formation in mice, rabbits, and multiple myeloma patients. Clin. Cancer Res. 10, 5949–5956 (2004).

  29. 29.

    Yu, K. & Ornitz, D. M. FGF signaling regulates mesenchymal differentiation and skeletal patterning along the limb bud proximodistal axis. Development 135, 483–491 (2008).

  30. 30.

    Petzold, G., Fischer, E. S. & Thoma, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase. Nature 532, 127–130 (2016).

  31. 31.

    Ramsey, M. R., He, L., Forster, N., Ory, B. & Ellisen, L. W. Physical association of HDAC1 and HDAC2 with p63 mediates transcriptional repression and tumor maintenance in squamous cell carcinoma. Cancer Res. 71, 4373–4379 (2011).

  32. 32.

    Hans, S., Liu, D. & Westerfield, M. Pax8 and Pax2a function synergistically in otic specification, downstream of the Foxi1 and Dlx3b transcription factors. Development 131, 5091–5102 (2004).

  33. 33.

    Saxena, A. & Cooper, K. L. Evolutionary biology: fin to limb within our grasp. Nature 537, 176–177 (2016).

  34. 34.

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

  35. 35.

    Yang, A. et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 397, 714–718 (1999).

  36. 36.

    Latina, A. et al. ΔNp63 targets cytoglobin to inhibit oxidative stress-induced apoptosis in keratinocytes and lung cancer. Oncogene 35, 1493–1503 (2016).

  37. 37.

    Wang, G. X. et al. ΔNp63 inhibits oxidative stress-induced cell death, including ferroptosis, and cooperates with the BCL-2 family to promote clonogenic survival. Cell Rep. 21, 2926–2939 (2017).

  38. 38.

    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).

  39. 39.

    Hansen, J. M., Harris, K. K., Philbert, M. A. & Harris, C. Thalidomide modulates nuclear redox status and preferentially depletes glutathione in rabbit versus rat limb. J. Pharmacol. Exp. Ther. 300, 768–776 (2002).

  40. 40.

    Sathyamurphy, A., Freund, S. M., Johnson, C. M., Allen, M. D. & Bycroft, M. Structural basis of p63α SAM domain mutants involved in AEC syndrome. FEBS J. 278, 2680–2688 (2011).

  41. 41.

    Zhao, X. et al. Zebrafish cul4a, but not cul4b, modulates cardiac and forelimb development by upregulating tbx5a expression. Hum. Mol. Genet. 24, 853–864 (2015).

  42. 42.

    Beedie, S. L. et al. In vivo screening and discovery of novel candidate thalidomide analogs in the zebrafish embryo and chicken embryo model systems. Oncotarget 7, 33237–33245 (2016).

  43. 43.

    Eichner, R. et al. Immunomodulatory drugs disrupt the cereblon–CD147–MCT1 axis to exert antitumor activity and teratogenicity. Nat. Med. 22, 735–743 (2016).

  44. 44.

    Lee, K. M. et al. Disruption of the cereblon gene enhances hepatic AMPK activity and prevents high-fat diet-induced obesity and insulin resistance in mice. Diabetes 62, 1855–1864 (2013).

  45. 45.

    Hagner, P. R. et al. CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL. Blood 126, 779–789 (2015).

  46. 46.

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

  47. 47.

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

  48. 48.

    Lai, A. C. & Crews, C. M. Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discov. 16, 101–114 (2017).

  49. 49.

    Thisse, C. & Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat. Protoc. 3, 59–69 (2008).

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Acknowledgements

We thank M. Manabe, M. Akiyama, S. Shoji and K. Taneda for technical assistance. We also thank A. J. Berk for critical comments on this manuscript. This work was supported by MEXT/JSPS KAKENHI grant numbers 17H06112 (to H.H. and Y.Y.), 15H04288 (to H.A.), 17H04213 (to T.I.), 17K14996 (to J.Y.) and 18H05502 (to T.I.). This work was also supported by MEXT-Supported Program for the Strategic Research Foundation at Private Universities S1411011 (to H.H.) and by PRESTO, JST JPMJPR1531 (to T.I.).

Author information

T.A.-O. performed most biochemical experiments corresponding to Fig. 1, Fig. 2, Fig. 3e, Fig. 5d, Supplementary Fig. 1, Supplementary Fig. 3 and Supplementary Fig. 6. M.D.S. performed biochemical experiments corresponding to Fig. 1. J.Y. performed biochemical experiments corresponding to Supplementary Fig. 3a. N.S. performed biochemical experiments corresponding to Supplementary Fig. 1g. H.A. and T.S. performed zebrafish experiments corresponding to Fig. 3, Fig. 4, Fig. 5, Supplementary Fig. 4, Supplementary Fig. 5 and Supplementary Fig. 7. T.A.-O., Y.Y., T.I., L.G. and H.H. interpreted all data. H.A. and K.A. interpreted zebrafish data. T.A.-O., T.I., L.G. and H.H. planned this study and wrote the manuscript. L.G. had the initial idea. L.G. and H.H. supervised the project. All authors discussed the results and approved the manuscript.

Correspondence to Luisa Guerrini or Hiroshi Handa.

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H.H. has received research support from Celgene Corporation.

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