Binding of Released Bim to Mcl-1 is a Mechanism of Intrinsic Resistance to ABT-199 which can be Overcome by Combination with Daunorubicin or Cytarabine in AML Cells.
Binding of Released Bim to Mcl-1 is a Mechanism of Intrinsic Resistance to ABT-199 which can be Overcome by Combination with Daunorubicin or Cytarabine in AML Cells.
复制标题
释放的 Bim 与 Mcl-1 的结合是 ABT-199 内在耐药性的机制，可通过与 AML 细胞中的柔红霉素或阿糖胞苷组合来克服
DOI：
10.1158/1078-0432.ccr-15-3057
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发表时间：
2016-09-01
期刊：
影响因子：
--
通讯作者：
Ge Y
中科院分区：
文献类型：
--
作者：
Niu X;Zhao J;Ma J;Xie C;Edwards H;Wang G;Caldwell JT;Xiang S;Zhang X;Chu R;Wang ZJ;Lin H;Taub JW;Ge Y
研究对ABT-199的固有耐药性的基础分子机制。
蛋白质印迹和实时RT-PCR用于单独或与细胞蛋白氨酸或daunorubicin结合使用ABT-199治疗后确定MCL-1的水平。 BIM和MCL-1的免疫沉淀用于确定ABT-199治疗对其与Bcl-2家族成员相互作用的影响。 BIM的慢病毒shRNA敲低和MCL-1的CRISPR敲低被用来确认它们在对ABT-199的抵抗中的作用。 JC-1分析和流式细胞仪用于确定药物诱导的凋亡。
BIM免疫沉淀在ABT-199治疗的细胞系和原发性患者样本中与Bcl-2的关联降低，但与MCL-1的关联增加而没有线粒体外膜电位的相应变化。 ABT-199治疗导致MCL-1蛋白水平升高，MCL-1转录物水平不变或降低，MCL-1蛋白半衰期增加，这表明与BIM的关联在稳定MCL-1蛋白中起作用。与单独的ABT-199相比，将常规化学治疗剂细胞或daunorubicin与ABT-199结合，导致DNA损伤增加以及MCL-1蛋白水平降低，以及AML细胞系中的细胞死亡的协同诱导，并从AML中获得的AML样品中的细胞死亡诱导诊断患者。
我们的结果表明，MCL-1固结BIM是一种内在的ABT-199耐药机制，并支持ABT-199与Cytarabine或Daunorubicin结合使用AML的临床发展。
Purpose: To investigate the molecular mechanism underlying intrinsic resistance to ABT-199. Experimental Design: Western blots and real-time RT-PCR were used to determine levels of Mcl-1 after ABT-199 treatment alone or in combination with cytarabine or daunorubicin. Immunoprecipitation of Bim and Mcl-1 were used to determine the effect of ABT-199 treatment on their interactions with Bcl-2 family members. Lentiviral short hairpin RNA knockdown of Bim and CRISPR knockdown of Mcl-1 were used to confirm their role in resistance to ABT-199. JC-1 assays and flow cytometry were used to determine drug-induced apoptosis. Results: Immunoprecipitation of Bim from ABT-199–treated cell lines and a primary patient sample demonstrated decreased association with Bcl-2, but increased association with Mcl-1 without corresponding change in mitochondrial outer membrane potential. ABT-199 treatment resulted in increased levels of Mcl-1 protein, unchanged or decreased Mcl-1 transcript levels, and increased Mcl-1 protein half-life, suggesting that the association with Bim plays a role in stabilizing Mcl-1 protein. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage along with decreased Mcl-1 protein levels, compared with ABT-199 alone, and synergistic induction of cell death in both AML cell lines and primary patient samples obtained from AML patients at diagnosis. Conclusions: Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance and supports the clinical development of ABT-199 in combination with cytarabine or daunorubicin for the treatment of AML. Clin Cancer Res; 22(17); 4440–51. ©2016 AACR.