Division of Hematology, Department of Medicine, Stanford University, Stanford, California.
Stanford Cancer Institute, Stanford University, Stanford, California.
Clin Cancer Res. 2016 Apr 15;22(8):1978-88. doi: 10.1158/1078-0432.CCR-15-1440. Epub 2015 Dec 3.
This study was performed to determine whether the investigational proteasome inhibitor ixazomib demonstrated selective antineoplastic activity against acute myelogenous leukemia cells expressing a mutated nucleophosmin-1 gene and to gain a better understanding of its mechanisms of action.
The cytotoxic effects of ixazomib treatment were analyzed in human acute myelogenous leukemia (AML) cell lines and primary AML samples expressing wild-type or mutated NPM1 (NPMc(+)). The potential roles of oxidative stress in mediating cytotoxic activity were determined using flow cytometry, enzyme-based assays, and Western blots.
Apoptosis induced by ixazomib was abrogated by knockdown of NPM1/NPMc(+)expression using an inducible shRNA construct and enhanced by NPMc(+)overexpression. Cytotoxicity was associated with superoxide generation and was reduced by the addition of the antioxidant N-acetylcysteine. AML cells expressing NPMc(+)had significantly reduced levels of intracellular glutathione and NADPH associated with reduced antioxidant responses to drug treatment. Treatment of 3 patients with relapsed NPMc(+)AML resulted in an antileukemic effect in 1 patient as demonstrated by a marked reduction of leukemic blasts in the peripheral blood. Efficacy was associated with superoxide generation, reduced glutathione levels, and reduced mRNA and protein expression of antioxidant effectors in responding cells.
In this study, a direct association was observed between NPMc(+)expression in AML, reduced antioxidant responses, and enhanced sensitivity to an oral proteasome inhibitor that induces oxidative stress. These data suggest that intracellular determinants of antioxidant responses may be good predictors of therapeutic response to ixazomib.
本研究旨在确定新型蛋白酶体抑制剂伊沙佐米对表达突变核磷蛋白-1（NPM1）基因的急性髓系白血病（AML）细胞是否具有选择性抗肿瘤活性，并深入了解其作用机制。
分析了伊沙佐米治疗对表达野生型或突变型 NPM1（NPMc(+)）的人急性髓系白血病（AML）细胞系和原代 AML 样本的细胞毒性作用。采用流式细胞术、酶法测定和 Western blot 确定氧化应激在介导细胞毒性活性中的潜在作用。
用诱导型 shRNA 构建体敲低 NPM1/NPMc(+)表达可阻断伊沙佐米诱导的凋亡，并增强 NPMc(+)过表达诱导的凋亡。细胞毒性与超氧自由基生成有关，抗氧化剂 N-乙酰半胱氨酸的加入可降低细胞毒性。表达 NPMc(+)的 AML 细胞的细胞内谷胱甘肽和 NADPH 水平显著降低，与药物治疗时抗氧化反应降低有关。对 3 例复发 NPMc(+)AML 患者进行治疗，1 例患者表现出明显的白血病细胞减少，外周血白血病细胞减少，表明有抗白血病作用。疗效与超氧自由基生成、谷胱甘肽水平降低以及反应细胞中抗氧化效应物的 mRNA 和蛋白表达减少有关。
在这项研究中，我们观察到 AML 中 NPMc(+)表达、抗氧化反应降低与口服蛋白酶体抑制剂（诱导氧化应激）敏感性增强之间存在直接关联。这些数据表明，细胞内抗氧化反应的决定因素可能是预测伊沙佐米治疗反应的良好指标。
