Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B‐cell lymphoma (DLBCL) tissues. Second mitochondria‐derived activator of caspases (Smac) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ‐mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pretreatment with the caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (zVAD.fmk) rescued BV6/CFZ‐induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9‐mediated NOXA inactivation inhibited BV6/CFZ‐induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies.

中文翻译：

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蛋白酶体抑制剂和Smac模拟物通过稳定NOXA和触发线粒体细胞凋亡，共同诱导弥漫性大B细胞淋巴瘤的细胞死亡。

在原发性弥漫性大B细胞淋巴瘤（DLBCL）组织中已鉴定出细胞凋亡蛋白（cIAP）1和cIAP2的细胞数量的拷贝数增加和表达水平的提高。半胱氨酸蛋白酶（Smac）模拟物的第二个线粒体衍生激活剂旨在拮抗IAP蛋白。但是，由于它们作为单药的作用有限，因此联合治疗代表了其临床发展的策略。因此，我们研究了Smac模拟BV6与蛋白酶体抑制剂的结合，并分析了其作用的分子机制。我们发现BV6处理可使DLBCL细胞对蛋白酶体抑制敏感。我们显示了通过BV6 / Carfilzomib（CFZ）处理的细胞活力和凋亡诱导的协同降低，这通过计算组合指数（CI）和Bliss得分得到证实。BV6和CFZ共同触发BAX和BAK的激活，从而促进细胞死亡，因为BAX和BAK的敲低显着降低了BV6 / CFZ介导的细胞死亡。BAX和BAK的激活伴随着线粒体膜电位（MMP）的丢失和胱天蛋白酶的激活。用半胱天冬酶抑制剂N-苄氧基羰基-Val-Ala-Asp-氟甲基酮（zVAD.fmk）进行预处理可挽救BV6 / CFZ诱导的细胞死亡，证实了半胱天冬酶的依赖性。单独使用CFZ或与BV6联合治疗会导致NOXA积累，这是细胞死亡所必需的，因为siRNA或成簇的规则间隔的短回文重复序列（CRISPR）/ Cas9介导的NOXA失活抑制了BV6 / CFZ诱导的细胞死亡。总之，这些实验表明BV6和CFZ协同诱导凋亡性细胞死亡通过线粒体途径。这些发现强调了Smac模拟物在使DLBCL细胞对蛋白酶体抑制敏感方面的作用，对进一步的（临床前）研究具有重要意义。