Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases

Abstract

Abstract Background 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). Methods 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. Results 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. Conclusion These results reveal that the bcr-abl gene disruption based on ZFNs may provide a treatment choice for imatinib resistant or intolerant CML patients.

Keywords

• Chronic myeloid leukemia
• Bcr-abl
• Zinc finger nucleases
• Homology-directed repair
• Oncogenicity