Role And Mechanism Of CaMKIIγ,a Critical Target Of The Natural Product Berbamine,in Regulating The Growth Of CML Cells

Background:Chronic myeloid leukemia (CML) accounts for approximately20%of all adult leukemias and significantly affects people’s health. CML is characterized by the presence of the Philadelphia chromosome (Ph+), which results from a chromosomal translocation between the Bcr gene on chromosome22and the Abl gene on chromosome9. This translocation produces the fusion protein Bcr-Abl that has constitutive kinase activity and actives multiple signaling pathways. These abnormal activations result in incontrollable cell proliferation and apoptosis tolerance, which ultimately induces the tumorigenesis of CML. Accordingly, tyrosine kinase inhibitors (TKIs, such as imatinib, IM) targeting Bcr-Abl has become a series of novel and attractive durgs in clinical setting. The five-years overall survival has increased to89.4%(86%-92%) in patients with CML after imatinib therapy. However, imatinib is not capable to kill all those cells with Bcr-Abl and simply effective in controlling CML at chronic phase, which partly attribute to TKI-resistance. Evidence indicates leukemia stem cell (LSCs) is one of the core mechanisms in TKI-resistance. Targeting this cluster of cells is a promising method in overcoming TKI-resistance.Berbamine (BBM) is a structurally unique bisbenzylisoquinoline isolated from TCM Berberis amurensis, and has been used in traditional Chinese medicine for treating a variety of diseases from inflammation to tumors for many years. Previous study of our group demonstrated that BBM and its derivatives potently inhibited the growth of imatinib (IM)-resistant CML cells but not normal hematopoietic cells. These results indicate BBM may significantly kill CML cells including those LSCs, which will provide a novel alternative way in CML therapy and curing this disease. And in the current study, we will study the role and mechanism of the berbamine target in regulating the growth of CML cells.Objects:1. To evaluate whether natural product BBM could efficiently eradicate LSCs or T315I mutant-Bcr-Abl cell clones of CML.2. To identify molecular targets of BBM for its antileukemia activity, and systematically evaluate the inhibitory effects of BBM on the candidate target.3. To investigate the potential molecular mechanism of the candidate target as a critical regulator for survive, proliferation and drug resistance of LSCs.Methods:1. We investigated the eradicative effect of BBM against imatinib (IM)-resistant-K562cells and T3151mutant-Bcr-Abl of CML through MTT in vitro.2. We evaluated the inhibitory effect of BBM on IM-resistance-cells K562xenografts in NOD/SCID mice by oral administration.3. We examined GSK3/Wnt/β-catenin and mitochondrial/Caspase-3expression level to reveal the apoptotic pathway.4. We used BBM as a molecular probe to identify molecular targets through compound structure-activity relationship (SAR) in vitro and in vivo, and computer docking modeling.5. We evaluated the inhibitory effect of BBM on the candidate target through immunoblot.6. We investigated the potential molecular mechanism of BBM against the candidate target through western blot and coimmunoprecipitation.Results:1. BBM significantly inhibited proliferations of both TKIs-resistant K562cells and KCL-22M cells with T315I mutant BCR-ABL in vitro.2. BBM eradicated IM-resistant K562leukemia xenografts (nude mice bearing IM-resistant K562xenografts) in vivo.3. BBM induced CML cell death by at least two manners:apoptosis and autophagy.4. BBM bound directly to the ATP-binding pocket of CaMKIIy and inhibited CaMKII y kinase activity in CML cells.5. CaMKIIy was the key molecular target of BBM in the CML cells.6. BBM killed the CML stem cells by inhibiting the NF-κB、β-catenin and Stat3pathways, which are CaMKII y dependent.Conclusions:1. BBM overrides TKI-resistance to LSCs and T315I mutant-Bcr-Abl of CML.2. BBM binds to and inhibits CaMK IIγ by targeting ATP binding pocket.3. CaMKIIγ is a critical target of berbamine for its antileukemia activity.4. Inhibitory activity of BBM on β-catenin, NF-κB, and Stat3networks can explain the anti-LSCs activity of BBM.