Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China.
J Exp Clin Cancer Res. 2018 Mar 20;37(1):62. doi: 10.1186/s13046-018-0732-4.
The bcr-abl fusion gene is the pathological origin of chronic myeloid leukemia (CML) and plays a critical role in the resistance of imatinib. Thus, bcr-abl disruption-based novel therapeutic strategy may warrant exploration. In our study, we were surprised to find that the characteristics of bcr-abl sequences met the design requirements of zinc finger nucleases (ZFNs).
We constructed the ZFNs targeting bcr-abl with high specificity through simple modular assembly approach. Western blotting was conducted to detect the expression of BCR-ABL and phosphorylation of its downstream STAT5, ERK and CRKL in CML cells. CCK8 assay, colony-forming assay and flow cytometry (FCM) were used to evaluate the effect of the ZFNs on the viablity and apoptosis of CML cells and CML CD34 cells. Moreover, mice model was used to determine the ability of ZFNs in disrupting the leukemogenesis of bcr-abl in vivo.
The ZFNs skillfully mediated 8-base NotI enzyme cutting site addition in bcr-abl gene of imatinib sensitive and resistant CML cells by homology-directed repair (HDR), which led to a stop codon and terminated the translation of BCR-ABL protein. As expected, the disruption of bcr-abl gene induced cell apoptosis and inhibited cell proliferation. Notably, we obtained similar result in CD34 cells from CML patients. Moreover, the ZFNs significantly reduced the oncogenicity of CML cells in mice.
These results reveal that the bcr-abl gene disruption based on ZFNs may provide a treatment choice for imatinib resistant or intolerant CML patients.
bcr-abl 融合基因是慢性髓性白血病（CML）的病理起源，在伊马替尼耐药中起关键作用。因此，基于 bcr-abl 断裂的新型治疗策略值得探索。在我们的研究中，我们惊讶地发现 bcr-abl 序列的特征符合锌指核酸酶（ZFNs）的设计要求。
我们通过简单的模块化组装方法构建了针对 bcr-abl 的高特异性 ZFNs。Western blot 检测 CML 细胞中 BCR-ABL 的表达及其下游 STAT5、ERK 和 CRKL 的磷酸化。CCK8 检测、集落形成实验和流式细胞术（FCM）用于评估 ZFNs 对 CML 细胞和 CML CD34 细胞活力和凋亡的影响。此外，还使用小鼠模型来确定 ZFNs 在体内破坏 bcr-abl 白血病发生的能力。
ZFNs 通过同源定向修复（HDR）巧妙地介导了 imatinib 敏感和耐药 CML 细胞中 bcr-abl 基因的 8 个碱基 NotI 酶切割位点的添加，导致一个终止密码子并终止了 BCR-ABL 蛋白的翻译。正如预期的那样，bcr-abl 基因的破坏诱导了细胞凋亡并抑制了细胞增殖。值得注意的是，我们在来自 CML 患者的 CD34 细胞中获得了类似的结果。此外，ZFNs 显著降低了小鼠中 CML 细胞的致癌性。
这些结果表明，基于 ZFNs 的 bcr-abl 基因断裂可能为伊马替尼耐药或不耐受的 CML 患者提供一种治疗选择。
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