Karabay Arzu Zeynep, Koc Asli, Ozkan Tulin, Hekmatshoar Yalda, Altinok Gunes Buket, Sunguroglu Asuman, Buyukbingol Zeliha, Atalay Arzu, Aktan Fugen
a Faculty of Pharmacy, Department of Biochemistry , Ankara University , Ankara , Turkey.
b Faculty of Medicine, Department of Medical Biology , Ankara University , Ankara , Turkey.
Hematology. 2018 Dec;23(10):765-770. doi: 10.1080/10245332.2018.1488795. Epub 2018 Jun 26.
Chronic myleoid leukemia (CML) is a myeloproliferative disorder characterized with the constitutive activation of Bcr-Abl tyrosine kinase which is a target for imatinib, the first line treatment option for CML. Constitutive activation of NFκB and β-catenin signaling promotes cellular proliferation and survival and resistance to Imatinib therapy in CML. Akirin-2 is a nuclear protein which is required for NFκB dependent gene expression as a cofactor and has been linked to Wnt/beta-catenin pathway. The purpose of this study is to examine Akirin-2, NFκB and β-catenin in imatinib resistance of CML and to test if any direct physical protein-protein interaction exists between NFkB and both β-catenin and Akirin-2.
RT-PCR and western blot were performed to determine Akirin-2, NFκB-p65 and β-catenin gene and protein expressions, Co-immunoprecipitation and chromatin immunoprecipitation analysis were carried out to detect the direct physical interactions and binding of NFκB-p65 and β-catenin proteins to MDR1 promoter region, respectively.
β-catenin and NFκB-p65 proteins bound to DNA promoter regions of MDR1 in imatinib-sensitive and resistant CML cells, whereas any direct protein-protein interaction could not be found between NFκB-p65 and Akirin-2 or β-catenin proteins. Nuclear β-catenin and NFκB-p65 levels increased in imatinib resistance. Moreover, increased Akirin-2 protein accumulation in the nucleus was shown for the first time in imatinib resistant CML cells.
We show for the first time that Akirin-2 can be a novel biomarker in imatinib resistance. Targeting Akirin-2, NFκB and β-catenin genes may provide an opportunity to overcome imatinib resistance in CML.
慢性髓性白血病（CML）是一种骨髓增殖性疾病，其特征在于Bcr-Abl酪氨酸激酶的组成性激活，该激酶是伊马替尼的作用靶点，伊马替尼是CML的一线治疗药物。NFκB和β-连环蛋白信号通路的组成性激活促进细胞增殖、存活以及CML对伊马替尼治疗的耐药性。Akirin-2是一种核蛋白，作为一种辅助因子，它是NFκB依赖性基因表达所必需的，并且与Wnt/β-连环蛋白通路有关。本研究的目的是检测CML对伊马替尼耐药性中的Akirin-2、NFκB和β-连环蛋白，并测试NFκB与β-连环蛋白和Akirin-2之间是否存在直接的物理性蛋白质-蛋白质相互作用。
进行逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法以确定Akirin-2、NFκB-p65和β-连环蛋白的基因及蛋白质表达，分别进行免疫共沉淀和染色质免疫沉淀分析以检测NFκB-p65和β-连环蛋白与多药耐药基因1（MDR1）启动子区域的直接物理相互作用及结合情况。
在伊马替尼敏感和耐药的CML细胞中，β-连环蛋白和NFκB-p65蛋白与MDR1的DNA启动子区域结合，然而在NFκB-p65与Akirin-2或β-连环蛋白之间未发现直接的蛋白质-蛋白质相互作用。在伊马替尼耐药时，核内β-连环蛋白和NFκB-p65水平升高。此外，首次在伊马替尼耐药的CML细胞中显示核内Akirin-2蛋白积累增加。
我们首次表明Akirin-2可能是伊马替尼耐药中的一种新型生物标志物。靶向Akirin-2、NFκB和β-连环蛋白基因可能为克服CML对伊马替尼的耐药性提供机会。
