Niu Xiaojia, Zhao Jianyun, Ma Jun, Xie Chengzhi, Edwards Holly, Wang Guan, Caldwell J Timothy, Xiang Shengyan, Zhang Xiaohong, Chu Roland, Wang Zhihong J, Lin Hai, Taub Jeffrey W, Ge Yubin
National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun, China. Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan.
National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun, China.
Clin Cancer Res. 2016 Sep 1;22(17):4440-51. doi: 10.1158/1078-0432.CCR-15-3057. Epub 2016 Apr 21.
To investigate the molecular mechanism underlying intrinsic resistance to ABT-199.
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.
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.
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.
研究对ABT - 199内在耐药的分子机制。
采用蛋白质免疫印迹法和实时逆转录聚合酶链反应来测定单独使用ABT - 199或与阿糖胞苷或柔红霉素联合使用后Mcl - 1的水平。对Bim和Mcl - 1进行免疫沉淀以确定ABT - 199处理对它们与Bcl - 2家族成员相互作用的影响。利用慢病毒短发夹RNA敲低Bim以及CRISPR敲低Mcl - 1来确认它们在对ABT - 199耐药中的作用。采用JC - 1检测法和流式细胞术来测定药物诱导的细胞凋亡。
对经ABT - 199处理的细胞系和一份原发性患者样本中的Bim进行免疫沉淀显示，与Bcl - 2的结合减少，但与Mcl - 1的结合增加，而线粒体外膜电位无相应变化。ABT - 199处理导致Mcl - 1蛋白水平升高、Mcl - 1转录水平不变或降低以及Mcl - 1蛋白半衰期延长，这表明与Bim的结合在稳定Mcl - 1蛋白中发挥作用。与单独使用ABT - 199相比，将传统化疗药物阿糖胞苷或柔红霉素与ABT - 199联合使用会导致DNA损伤增加以及Mcl - 1蛋白水平降低，并在急性髓系白血病细胞系和从诊断时的急性髓系白血病患者获取的原发性患者样本中协同诱导细胞死亡。
我们的结果表明，Mcl - 1对Bim的隔离是对ABT - 199内在耐药的一种机制，并支持ABT - 199与阿糖胞苷或柔红霉素联合用于治疗急性髓系白血病的临床开发。临床癌症研究；22(17)；4440 - 51。©2016美国癌症研究协会。
