Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4CRBN activity
Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4CRBN activity
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DOI：
10.1182/blood-2018-01-821769
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发表时间：
2018-09-20
期刊：
影响因子：
20.3
通讯作者：
Ebert, Benjamin L.
中科院分区：
文献类型：
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作者：
Sievers, Quinlan L.;Gasser, Jessica A.;Ebert, Benjamin L.
来那度胺通过促进Ikaros家族锌指蛋白1（IKZF 1）、IKZF 3和酪蛋白激酶1 α（CK 1 α）与cereblon（CRBN）（CRL 4（CRBN）E3泛素连接酶的底物受体）的相互作用，介导Ikaros家族锌指蛋白1（IKZF 1）、IKZF 3和酪蛋白激酶1 α（CK 1 α）的泛素化和降解。通过这种机制，来那度胺是多发性骨髓瘤和伴有染色体5 q缺失的骨髓增生异常综合征（MDS）[del（5 q）MDS]的临床有效治疗药物。为了鉴定来那度胺诱导的CRL 4（CRBN）活性所需的细胞机制，我们在来那度胺敏感的骨髓瘤细胞系中进行了阳性选择、基因组规模的规律间隔成簇短回文重复序列（CRISPR）-CRISPR相关蛋白9（Cas9）筛选。CRBN是排名最高的基因，所有CRBN靶向向导RNA（gRNA）排名为6个得分最高的gRNA。使用IKZF 3降解决定子报告基因测定来那度胺诱导的蛋白质降解的反筛选强调了cullin-RING连接酶neddylation和2种E2泛素缀合酶的调节剂是来那度胺有效诱导的蛋白质降解所必需的。我们证明了UBE 2 M或组成型光形态发生9（COP 9）信号体成员的缺失导致cullin 4A的neddylation改变，并损害来那度胺依赖性CRL 4（CRBN）活性。此外，我们确定UBE 2D 3和UBE 2G 1在通过CRL 4（CRBN）的底物泛素化中发挥不同的作用，其中UBE 2D 3通过单泛素化作用于引发靶点，而UBE 2G 1用于延伸具有赖氨酸48键的聚泛素链。UBE 2D 3和UBE 2G 1的验证突出了CRISPR-Cas9筛选鉴定E2泛素缀合酶和E3泛素连接酶复合物配对的功能能力。更广泛地说，这些发现确立了骨髓瘤中来那度胺依赖性CRL 4（CRBN）功能所需的关键蛋白质，并为潜在的耐药机制提供了信息。
Lenalidomide mediates the ubiquitination and degradation of Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and casein kinase 1 alpha (CK1 alpha) by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4(CRBN) 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(CRBN) 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. CRBN was the top-ranking gene, with all CRBN-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(CRBN) activity. Additionally, we established that UBE2D3 and UBE2G1 play distinct roles in substrate ubiquitination by CRL4(CRBN), 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(CRBN) function in myeloma and inform potential mechanisms of drug resistance.