CC-90009 is a novel cereblon E3 ligase modulator (CELMoD) currently under investigation in a phase I clinical study in relapsed or refractory acute myeloid leukemia (R/R AML) (CC-90009-AML-001; NCT02848001). CC-90009 coopts the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex to target the translation termination factor G1 to S phase transition 1 (GSPT1) for ubiquitination and proteasomal degradation, resulting in rapid induction of apoptosis and growth inhibition in AML cell lines and primary patient blasts. To further elucidate the mechanism of action of CC-90009 in AML, we performed a genome-wide CRISPR/Cas9 screen to identify gene(s) whose knockout abrogate(s) the response to CC-90009 in a sensitive AML cell line. In addition to well-established key regulatory proteins required for the activity of all known cereblon modulators, which include components of the CRL4CRBN complex, E2 ubiquitin conjugating enzymes UBE2G1 and UBE2D3, and members of the neddylation and deneddylation machinery, interestingly, the screen identified the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreased the production of full-length CRBN transcript via modulating alternative splicing of CRBN mRNA, leading to significant downregulation of cereblon expression and hence diminished response to CC-90009. The screen also revealed that mTOR signaling and the integrated stress response (ISR) specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Since CC-90009 inhibits protein translation, it is reasonable to expect interactions with regulators of this pathway. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 , at least in part by reducing CC-90009 induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Loss of GCN2 significantly attenuated the growth inhibitory effect of CC-90009, and this effect can be rescued with GCN2 wild-type but not enzymatically-dead mutants. Collectively, the antitumor activity of CC-90009, a first-in-class GSPT1 degrader, in AML cell lines is mediated by multiple layers of signaling networks and machinery, the elucidation of which reveals the underlying mechanism by which CC-90009 exerts its anti-AML activity and informs on the pathways for further study of CC-90009's clinical utility. Disclosures Lu: Celgene Corporation: Employment, Equity Ownership. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Jang:Celgene: Employment, Equity Ownership. Wang:Celgene: Employment, Equity Ownership. Rolfe:Celgene: Employment, Equity Ownership.

中文翻译：

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通过全基因组 CRISPR 筛选阐明 CC-90009（一种新型 Cereblon E3 连接酶调节剂）在 AML 中的作用机制

CC-90009 是一种新型的 cereblon E3 连接酶调节剂 (CELMoD)，目前正在对复发或难治性急性髓性白血病 (R/R AML) 进行 I 期临床研究 (CC-90009-AML-001; NCT02848001)。CC-90009 采用 CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 泛素连接酶复合物靶向翻译终止因子 G1 至 S 相变 1 (GSPT1) 进行泛素化和蛋白酶体降解，从而快速诱导细胞凋亡和生长抑制AML 细胞系和原发性患者原始细胞。为了进一步阐明 CC-90009 在 AML 中的作用机制，我们进行了全基因组 CRISPR/Cas9 筛选，以鉴定基因敲除消除敏感 AML 细胞系中对 CC-90009 的反应的基因。除了所有已知的 cereblon 调节剂（包括 CRL4CRBN 复合物的组分、E2 泛素结合酶 UBE2G1 和 UBE2D3 以及 neddylation 和 deneddylation 机制的成员）的活性所需的成熟的关键调节蛋白之外，有趣的是，筛选确定了ILF2 和 ILF3 异二聚体复合物作为 cereblon 表达的新调节剂。敲除 ILF2/ILF3 通过调节 CRBN mRNA 的可变剪接减少了全长 CRBN 转录物的产生，导致 cereblon 表达显着下调，从而降低了对 CC-90009 的反应。筛选还显示，与其他 cereblon 调节剂相比，mTOR 信号传导和综合应激反应 (ISR) 专门调节对 CC-90009 的反应。由于 CC-90009 抑制蛋白质翻译，期望与该途径的监管机构相互作用是合理的。通过灭活 TSC1 和 TSC2 使 mTOR 通路过度活化，至少部分通过减少 CC-90009 诱导的 GSPT1 与 cereblon 的结合和随后的 GSPT1 降解来保护免受 CC-90009 的生长抑制作用。另一方面，GSPT1 降解促进了 AML 细胞中 GCN1/GCN2/ATF4 通路的激活和随后的细胞凋亡。GCN2 的缺失显着减弱了 CC-90009 的生长抑制作用，这种作用可以通过 GCN2 野生型而非酶促死亡突变体来挽救。总的来说，CC-90009（一种一流的 GSPT1 降解剂）在 AML 细胞系中的抗肿瘤活性是由多层信号网络和机制介导的，其阐明揭示了 CC-90009 发挥其抗 AML 活性的潜在机制，并为进一步研究 CC-90009 的临床效用提供了信息。披露 Lu：Celgene Corporation：雇佣、股权。Surka:Celgene：就业、股权。Lu:Celgene Corporation：就业、股权。Jang:Celgene：就业、股权。王：Celgene：就业，股权。Rolfe:Celgene：就业、股权。股权。Jang:Celgene：就业、股权。王：Celgene：就业，股权。Rolfe:Celgene：就业、股权。股权。Jang:Celgene：就业、股权。王：Celgene：就业，股权。Rolfe:Celgene：就业、股权。