|Publication Type||Journal Article|
|Year of Publication||2018|
|Authors||Sievers, QL, Gasser, JA, Cowley, GS, Fischer, ES, Ebert, BL|
|Date Published||2018 Jul 24|
Lenalidomide mediates the ubiquitination and degradation of IKZF1, IKZF3 and CK1α by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4 E3 ubiquitin ligase. Through this mechanism, lenalidomide is a clinically effective treatment for multiple myeloma and myelodysplastic syndrome with deletion of chromosome 5q (del(5q) MDS). To identify the cellular machinery required for lenalidomide-induced CRL4 activity, we performed a positive selection, genome-scale CRISPR-Cas9 screen in a lenalidomide-sensitive myeloma cell line. CRBN was the top-ranking gene, with all CRBN-targeting gRNAs ranking as the 6 highest-scoring gRNAs. A counter-screen using an IKZF3 degron reporter to assay lenalidomide-induced protein degradation highlighted regulators of cullin-RING ligase neddylation and two E2 ubiquitin-conjugating enzymes as necessary for efficient lenalidomide-induced protein degradation. We demonstrated that loss of UBE2M or members of the COP9 signalosome results in altered neddylation of cullin 4A and impairs lenalidomide-dependent CRL4 activity. Additionally, we established that UBE2D3 and UBE2G1 play distinct roles in substrate ubiquitination by CRL4, with UBE2D3 acting to prime targets via monoubiquitination and UBE2G1 functioning to extend polyubiquitin chains with lysine 48 linkages. The validation of UBE2D3 and UBE2G1 highlights the functional capacity of CRISPR-Cas9 screening to identify E2 ubiquitin conjugating enzyme and E3 ubiquitin ligase complex pairings. More broadly, these findings establish key proteins required for lenalidomide-dependent CRL4 function in myeloma and inform potential mechanisms of drug resistance.