The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.

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.

Recent discoveries suggest that the critical events leading to the anti-proliferative activity of the IMiD immunomodulatory agents lenalidomide and pomalidomide in multiple myeloma (MM) cells are initiated by Cereblon-dependent ubiquitination and proteasomal degradation of substrate proteins Ikaros (IKZF1) and Aiolos (IKZF3). By performing kinetic analyses, we found that the downregulation or proteasomal degradation of Ikaros and Aiolos led to specific and sequential downregulation of c-Myc followed by IRF4 and subsequent growth inhibition and apoptosis. Notably, to ensure growth inhibition and cell death, sustained downregulation of Ikaros and Aiolos, c-Myc or IRF4 expression was required. In addition, we found that the half-maximal rate, rather than the final extent of Ikaros and Aiolos degradation, correlated to the relative efficacy of growth inhibition by lenalidomide or pomalidomide. Finally, we observed that all four transcription factors were elevated in primary MM samples compared with normal plasma cells. Taken together, our results suggest a functional link between Ikaros and Aiolos, and the pathological dysregulation of c-Myc and IRF4, and provide a new mechanistic understanding of the relative efficacy of lenalidomide and pomalidomide based on the kinetics of substrate degradation and downregulation of their downstream targets.

Key Points Pomalidomide leads to rapid immune activation in vivo correlating with clinical outcome in relapsed myeloma. Baseline expression of ikaros/aiolos protein in tumor cells is not predictive of outcome.

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