蛋白酶体抑制剂已用于治疗复发性多发性骨髓瘤和套细胞淋巴瘤。观察到的由蛋白酶体抑制剂引起的毒性是许多具有不同敏感性的癌细胞的普遍表型。在这项研究中，我们利用基因编辑方法研究了蛋白酶体抑制剂硼替佐米毒性的保守机制。我们利用不同的 caspase 敲除细胞的研究结果表明，硼替佐米通过激活 caspase-9 和 caspase-3/7 诱导经典的内源性细胞凋亡，导致成孔蛋白 GSDME 裂解和随后的裂解性细胞死亡或称为继发性坏死，这种表型也在许多细胞凋亡触发因素，如 HeLa 细胞中的 TNFα 加 CHX、DTT 和衣霉素治疗。此外，通过敲除几乎所有 BH3-only 蛋白，包括 BIM、BAD、BID、BMF 和 PUMA，我们证明 NOXA 是唯一负责硼替佐米诱导细胞凋亡的 BH3-only 蛋白。值得注意的是，NOXA 因选择性结合 MCL-1 和 A1 而闻名，但我们利用不同的 BH3 模拟物以及免疫沉淀测定的研究表明，除了 NOXA 与 MCL-1 的组成型相互作用外，硼替佐米后 NOXA 的积累治疗使其与 BCL-XL 相互作用，然后同时缓解抗凋亡蛋白 BCL-XL 和 MCL-1 对细胞凋亡的抑制。此外，虽然研究中观察到硼替佐米诱导显着的内质网应激和JNK激活，但进一步的基因耗竭实验证明，硼替佐米诱导的细胞凋亡的发生独立于内质网应激相关的凋亡因子CHOP和JNK。总之，这些结果提供了关于 NOXA 在硼替佐米诱导的细胞凋亡中除 MCL-1 之外的 BCL-XL 失活中的关键作用的可靠结论。版权所有 © 2024 Elsevier B.V. 保留所有权利。

Proteasome inhibitors have been employed in the treatment of relapsed multiple myeloma and mantle cell lymphoma. The observed toxicity caused by proteasome inhibitors is a universal phenotype in numerous cancer cells with different sensitivity. In this study, we investigate the conserved mechanisms underlying the toxicity of the proteasome inhibitor bortezomib using gene editing approaches. Our findings utilizing different caspase knocking out cells reveal that bortezomib induces classic intrinsic apoptosis by activating caspase-9 and caspase-3/7, leading to pore-forming protein GSDME cleavage and subsequent lytic cell death or called secondary necrosis, a phenotype also observed in many apoptosis triggers like TNFα plus CHX, DTT and tunicamycin treatment in HeLa cells. Furthermore, through knocking out of nearly all BH3-only proteins including BIM, BAD, BID, BMF and PUMA, we demonstrate that NOXA is the sole BH3-only protein responsible for bortezomib-induced apoptosis. Of note, NOXA is well known for selectively binding to MCL-1 and A1, but our studies utilizing different BH3 mimetics as well as immunoprecipitation assays indicate that, except for the constitutive interaction of NOXA with MCL-1, the accumulation of NOXA after bortezomib treatment allows it to interact with BCL-XL, then simultaneous relieving suppression on apoptosis by both anti-apoptotic proteins BCL-XL and MCL-1. In addition, though bortezomib-induced significant ER stress and JNK activation were observed in the study, further genetic depletion experiments prove that bortezomib-induced apoptosis occurs independently of ER stress-related apoptosis factor CHOP and JNK. In summary, these results provide a solid conclusion about the critical role of NOXA in inactivation of BCL-XL except MCL-1 in bortezomib-induced apoptosis.Copyright © 2024 Elsevier B.V. All rights reserved.