shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance
shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance
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DOI：
10.1182/blood-2014-08-588855
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发表时间：
2015-03-12
期刊：
影响因子：
20.3
通讯作者：
Deininger, Michael W.
中科院分区：
文献类型：
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作者：
Khorashad, Jamshid S.;Eiring, Anna M.;Deininger, Michael W.
缺乏BCR-ABL1激活域突变的慢性髓系白血病(CML)患者对酪氨酸激酶抑制物(TKI)耐药的机制尚不完全清楚。为了确定与BCR-ABL1激酶活性无关的TKI耐药机制，我们将一个靶向5000个细胞信号基因的慢病毒短发夹状RNA(ShRNA)文库导入K562(R)，K562(R)是一种对BCR-ABL1激酶不依赖的CML细胞株，仅表达天然BCR-ABL1。一种定制的算法识别了与TKI敏感的对照相比，shRNA介导的敲除明显损害K562(R)细胞生长的基因。核质运输复合体的两个组分RAN和XPO1(CRM1)是最热门的候选基因。ShRNA介导的RAN抑制或用XPO1抑制剂KPT-330(Selinexor)处理细胞，增加了具有激酶非依赖性TKI耐药的CML细胞系对伊马替尼的敏感性。在伊马替尼存在下，RAN或XPO1抑制新诊断和TKI耐药CML患者CD34(+)细胞的集落形成，而对正常脐血或携带BCR-ABL1(T315I)突变的患者的CD34(+)细胞没有影响。这些数据表明RAN与BCR-ABL1激酶非依赖性伊马替尼耐药有关，并表明shRNA文库筛选有助于识别CML耐药的关键途径。
The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting similar to 5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.