SummaryLenalidomide 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 CRL4CRBN E3 ubiquitin ligase, 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 lenalidomide's therapeutic window 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.

In historical attempts to treat morning sickness, use of the drug thalidomide led to the birth of thousands of children with severe birth defects. Despite their teratogenicity, thalidomide and related IMiD drugs are now a mainstay of cancer treatment; however, the molecular basis underlying the pleiotropic biology and characteristic birth defects remains unknown. Here we show that IMiDs disrupt a broad transcriptional network through induced degradation of several C2H2 zinc finger transcription factors, including SALL4, a member of the spalt-like family of developmental transcription factors. Strikingly, heterozygous loss of function mutations in SALL4 result in a human developmental condition that phenocopies thalidomide-induced birth defects such as absence of thumbs, phocomelia, defects in ear and eye development, and congenital heart disease. We find that thalidomide induces degradation of SALL4 exclusively in humans, primates, and rabbits, but not in rodents or fish, providing a mechanistic link for the species-specific pathogenesis of thalidomide syndrome.

Molecular glue compounds induce protein-protein interactions that, inthe context of a ubiquitin ligase, lead to protein degradation. 1 Unlike traditional enzyme inhibitors, such molecular glue degraders act sub-stoichiometrically to catalyse rapid depletion of previously inaccessible targets. 2 They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Through systematic mining of databases for correlations between the cytotoxicity of 4,518 clinical and pre-clinical small molecules and E3 ligase expression levels across hundreds of human cancer cell lines, 3-5 we identified CR8, a cyclin-dependent kinase (CDK) inhibitor, 6 as a compound that acts as a molecular glue degrader. A solvent-exposed pyridyl moiety of CR8, in its CDKbound form, induces CDK12-cyclin K complex formation with DDB1, the CUL4 adaptor protein, bypassing the requirement for a substrate receptor and presenting cyclin K (cycK) for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy to turn target binders into molecular glues.

Lenalidomide acts by a novel drug mechanism—modulation of the substrate specificity of the CRL4CRBN E3 ubiquitin ligase. In multiple myeloma, lenalidomide induces the ubiquitination of IKZF1 and IKZF3 by CRL4CRBN. Subsequent proteasomal degradation of these transcription factors kills multiple myeloma cells. In del(5q) myelodysplastic syndrome, lenalidomide induces the degradation of CK1α, which preferentially affects del(5q) cells because they express this gene at haploinsufficient levels. In the future, modulation of ubiquitin ligase function may enable us to target previously “undruggable” proteins.