Thalidomide Teratogenicity Target: In the late 1950s and early 1960s, thalidomide was prescribed to pregnant women as a cure for morning sickness, but it was then found to have developmental defects, most obviously, stunted limbs in thousands of babies. Although its use was banned worldwide, thalidomide has since been found to be a valuable treatment for a range of cancers, inflammatory disorders, and leprosy. Several hypotheses have been proposed, but the mechanism of action of thalidomide is unknown. Using zebrafish and chicken as animal models, Ito et al. (p. 1345 ) show that the protein cereblon is a primary target of thalidomide. Thalidomide exerts teratogenic effects by binding to cereblon and inhibiting associated enzymatic activity important for limb development. Knowing the mechanism of action of thalidomide should encourage the search for thalidomide derivatives without teratogenic activity.

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN–DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN^YW/AA. Overexpression of CRBN wild-type protein, but not CRBN^YW/AA mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21^WAF-1 expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

Summary: Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4^CRBN. T cell co‐stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4^CRBN substrates responsible for T cell co‐stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4^CRBN is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4^CRBN with enhanced ubiquitination leading to cereblon‐dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin‐2 expression. The findings link lenalidomide‐ or pomalidomide‐induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4^CRBN, leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

Progress in strategies aimed at breaking down therapeutic target proteins has led to a paradigm shift in drug discovery. Thalidomide and its derivatives are the only protein degraders currently used in clinical practice. Our understanding of the molecular mechanism of action of thalidomide and its derivatives has advanced dramatically since the identification of cereblon (CRBN) as their direct target. The binding of thalidomide derivatives to CRBN, a substrate recognition receptor for Cullin 4 RING E3 ubiquitin ligase (CRL4), induces the recruitment of non-native substrates to CRL4^CRBN and their subsequent degradation. This discovery was a breakthrough in the current rapid development of protein-degrading agents because clarification of the mechanism of action of thalidomide derivatives has demonstrated the clinical value of these compounds. This review provides an overview of the mechanism of action of thalidomide and its derivatives and describes perspectives for protein degraders.