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Blood DOI:10.1182/blood-2018-01-821769

Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4 activity.

Publication TypeJournal Article
Year of Publication2018
AuthorsSievers, QL, Gasser, JA, Cowley, GS, Fischer, ES, Ebert, BL
JournalBlood
Volume132
Issue12
Pages1293-1303
Date Published2018 09 20
ISSN1528-0020
KeywordsAntineoplastic Agents, Cell Line, Tumor, CRISPR-Cas Systems, Drug Resistance, Neoplasm, Humans, Lenalidomide, Multiple Myeloma, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitination
Abstract

Lenalidomide mediates the ubiquitination and degradation of Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and casein kinase 1α (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 of multiple myeloma and myelodysplastic syndrome (MDS) 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 clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) screen in a lenalidomide-sensitive myeloma cell line. was the top-ranking gene, with all -targeting guide RNAs (gRNAs) ranking as the 6 highest-scoring gRNAs. A counterscreen using an IKZF3 degron reporter to assay lenalidomide-induced protein degradation highlighted regulators of cullin-RING ligase neddylation and 2 E2 ubiquitin-conjugating enzymes as necessary for efficient lenalidomide-induced protein degradation. We demonstrated that loss of UBE2M or members of the constitutive photomorphogenesis 9 (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.

DOI10.1182/blood-2018-01-821769
Pubmed

https://www.ncbi.nlm.nih.gov/pubmed/30042095?dopt=Abstract

Alternate JournalBlood
PubMed ID30042095
PubMed Central IDPMC6148446
Grant ListP50 CA206963 / CA / NCI NIH HHS / United States
R01 HL082945 / HL / NHLBI NIH HHS / United States
T32 GM007753 / GM / NIGMS NIH HHS / United States
P01 CA066996 / CA / NCI NIH HHS / United States
R01 CA214608 / CA / NCI NIH HHS / United States
P30 CA006516 / CA / NCI NIH HHS / United States
/ HHMI / Howard Hughes Medical Institute / United States