Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL.
Blood Adv. 2019 Jul 23;3(14):2105-2117. doi: 10.1182/bloodadvances.2019031732.
Genome-wide CRISPR/Cas9 screens represent a powerful approach to studying mechanisms of drug action and resistance. Cereblon modulating agents (CMs) have recently emerged as candidates for therapeutic intervention in primary effusion lymphoma (PEL), a highly aggressive cancer caused by Kaposi's sarcoma-associated herpesvirus. CMs bind to cereblon (CRBN), the substrate receptor of the cullin-RING type E3 ubiquitin ligase CRL4, and thereby trigger the acquisition and proteasomal degradation of neosubstrates. Downstream mechanisms of CM toxicity are incompletely understood, however. To identify novel CM effectors and mechanisms of CM resistance, we performed positive selection CRISPR screens using 3 CMs with increasing toxicity in PEL: lenalidomide (LEN), pomalidomide (POM), and CC-122. Results identified several novel modulators of the activity of CRL4 The number of genes whose inactivation confers resistance decreases with increasing CM efficacy. Only inactivation of CRBN conferred complete resistance to CC-122. Inactivation of the E2 ubiquitin conjugating enzyme UBE2G1 also conferred robust resistance against LEN and POM. Inactivation of additional genes, including the Nedd8-specific protease SENP8, conferred resistance to only LEN. SENP8 inactivation indirectly increased levels of unneddylated CUL4A/B, which limits CRL4 activity in a dominant negative manner. Accordingly, sensitivity of SENP8-inactivated cells to LEN is restored by overexpression of CRBN. In sum, our screens identify several novel players in CRL4 function and define pathways to CM resistance in PEL. These results provide rationale for increasing CM efficacy on patient relapse from a less-efficient CM. Identified genes could finally be developed as biomarkers to predict CM efficacy in PEL and other cancers.
全基因组 CRISPR/Cas9 筛选代表了一种研究药物作用和耐药机制的强大方法。 cereblon 调节因子（CMs）最近被认为是治疗原发性渗出性淋巴瘤（PEL）的候选药物，PEL 是一种由卡波西肉瘤相关疱疹病毒引起的高度侵袭性癌症。 CMs 与 cereblon（CRBN）结合，CRBN 是 cullin-RING 型 E3 泛素连接酶 CRL4 的底物受体，从而触发新底物的获得和蛋白酶体降解。然而，CM 毒性的下游机制尚不完全清楚。为了确定新型 CM 效应物和 CM 耐药机制，我们使用三种在 PEL 中具有逐渐增加毒性的 CMs（来那度胺[LEN]、泊马度胺[POM]和 CC-122）进行正选择 CRISPR 筛选。结果确定了几种新的 CRL4 活性调节剂，随着 CM 功效的增加，其失活赋予耐药性的基因数量减少。只有 CRBN 的失活赋予对 CC-122 的完全耐药性。E2 泛素连接酶 UBE2G1 的失活也赋予对 LEN 和 POM 的强大耐药性。此外，包括 Nedd8 特异性蛋白酶 SENP8 在内的其他基因的失活仅赋予对 LEN 的耐药性。SENP8 的失活间接增加了未缀合的 CUL4A/B 的水平，这以显性负性方式限制了 CRL4 的活性。因此，SENP8 失活细胞对 LEN 的敏感性通过 CRBN 的过表达得到恢复。总之，我们的筛选确定了 CRL4 功能的几个新参与者，并定义了 PEL 中 CM 耐药的途径。这些结果为从效率较低的 CM 复发的患者增加 CM 功效提供了依据。鉴定的基因最终可作为预测 PEL 和其他癌症中 CM 功效的生物标志物。
