Genome-Scale Screen Reveals Genes Required for Lenalidomide-Mediated Degradation of Aiolos By CRL4-CRBN

Lenalidomide exerts its therapeutic effects in the malignancy multiple myeloma by facilitating the degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by the CRL4-CRBN E3 ubiquitin ligase. In the following study we utilized a positive selection, genome-scale CRISPR-Cas9 screen in the lenalidomide-sensitive myeloma cell line, MM1S, to further our understanding of the molecular machinery which regulates and is required for lenalidomide-mediated modulation of CRL4-CRBN. The gRNAs demonstrating the greatest enrichment following selection with lenalidomide belong to cereblon, the molecular target of lenalidomide and substrate receptor for the CRL4-CRBN ubiquitin ligase. Additionally, 20 of the top 30 genes highlighted by the screen are implicated in both the positive and negative regulation of cullin-ring ligases (CRLs), emphasizing both the importance of cereblon's E3 ligase function in mediating cell death as well as the equilibrium of forces required for proper function of CRLs. Among these genes were the ubiquitin-donor E2 enzymes UBE2D3 and UBE2G1; despite the relevance of E2s to E3 ligase biology it has been difficult to determine via traditional methods which of the ~35 E2 enzymes are utilized by a given E3 ligase. Here we demonstrate that G1 and D3 fulfill distinct functional roles and cooperate with the CRL4CRBN E3 ligase to ubiquitinate an aiolos reprorter with lysine-48 linkages. In aggregate these findings confirm the power of CRISPR-Cas9 positive selection screening to reveal drug mechanism-of-action and provides a paradigm for the identification and functional characterization of E2-E3 pairings.