Pevonedistat (MLN4924), a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit (NAE1), has demonstrated significant therapeutic potential in several malignancies. Although multiple mechanisms-of-action have been identified, how MLN4924 induces cell death and its potential as a combinatorial agent with standard-of-care (SoC) chemotherapy in colorectal cancer (CRC) remains largely undefined. In an effort to understand MLN4924-induced cell death in CRC, we identified p53 as an important mediator of the apoptotic response to MLN4924. We also identified roles for the extrinsic (TRAIL-R2/caspase-8) and intrinsic (BAX/BAK) apoptotic pathways in mediating the apoptotic effects of MLN4924 in CRC cells, as well as a role for BID, which modulates a cross-talk between these pathways. Depletion of the anti-apoptotic protein FLIP, which we identify as a novel mediator of resistance to MLN4924, enhanced apoptosis in a p53-, TRAIL-R2/DR5-, and caspase-8-dependent manner. Notably, TRAIL-R2 was involved in potentiating the apoptotic response to MLN4924 in the absence of FLIP, in a ligand-independent manner. Moreoever, when paired with SoC chemotherapies, MLN4924 demonstrated synergy with the irinotecan metabolite SN38. The cell death induced by MLN4924/SN38 combination was dependent on activation of mitochondria through BAX/BAK, but in a p53-independent manner, an important observation given the high frequency of TP53 mutation(s) in advanced CRC. These results uncover mechanisms of cell death induced by MLN4924 and suggest that this second-generation proteostasis-disrupting agent may have its most widespread activity in CRC, in combination with irinotecan-containing treatment regimens.

Pevonedistat (MLN4924) 是 NEDD8 激活酶 E1 调节亚基 (NAE1) 的选择性抑制剂，已在多种恶性肿瘤中表现出显着的治疗潜力。尽管已经确定了多种作用机制，但 MLN4924 如何诱导细胞死亡及其作为结直肠癌 (CRC) 护理标准 (SoC) 化疗的组合药物的潜力仍然很大程度上未明确。为了了解 MLN4924 诱导的 CRC 细胞死亡，我们确定 p53 是 MLN4924 细胞凋亡反应的重要介质。我们还确定了外在（TRAIL-R2/caspase-8）和内在（BAX/BAK）凋亡途径在介导 MLN4924 在 CRC 细胞中的凋亡作用中的作用，以及调节串扰的 BID 的作用在这些路径之间。抗凋亡蛋白 FLIP（我们将其确定为 MLN4924 抗性的新型介质）的耗尽，以 p53、TRAIL-R2/DR5 和 caspase-8 依赖性方式增强细胞凋亡。值得注意的是，在 FLIP 不存在的情况下，TRAIL-R2 以不依赖配体的方式参与增强对 MLN4924 的细胞凋亡反应。此外，当与 SoC 化疗药物配合使用时，MLN4924 表现出与伊立替康代谢物 SN38 的协同作用。MLN4924/SN38组合诱导的细胞死亡依赖于BAX/BAK对线粒体的激活，但以不依赖于p53的方式，鉴于晚期CRC中TP53突变的高频率，这是一个重要的观察结果。这些结果揭示了 MLN4924 诱导的细胞死亡机制，并表明这种第二代蛋白质稳态破坏剂与含伊立替康的治疗方案相结合，可能在结直肠癌中具有最广泛的活性。