Localisation, activity and targeting of Bcr-Abl in chronic myeloid leukaemia.
PhD thesis, University of Glasgow.
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Chronic myeloid leukaemia (CML) is a myeloproliferative disease of stem cell origin. It is characterised by the Philadelphia chromosome and Bcr-Abl oncoprotein which is a constitutively activated tyrosine kinase that is causative in CML. Treatment of CML was revolutionized by tyrosine kinase inhibitors (TKIs) in the last decade. TKIs target Bcr-Abl and block its tyrosine kinase activity. Despite the success of TKI in eliminating differentiated CML cells, primitive quiescent CML stem cells still resist or persist with TKI treatment. In this study, several strategies have been investigated towards elimination of TKI-insensitive primitive CML stem cells.
At first, the effect of nuclear entrapment of Bcr-Abl protein in CML cells was studied. Although Bcr-Abl protein is located in the cytoplasm of CML cells, TKIs, such as imatinib mesylate (IM), can induce the nuclear translocation of Bcr-Abl. Nuclear Bcr-Abl tyrosine kinase is capable of inducing apoptosis after drug washout, and leptomycin B (LMB) can trap translocated Bcr-Abl in the nucleus. Primary CML CD34+ cells were treated with IM and LMB either continuously or sequentially. It was found that neither regimen significantly increased the anti-proliferative effect of IM in primary CML CD34+ cells. It was also observed that the majority of Bcr-Abl was still retained in the cytoplasm of CML cells treated with IM and LMB, suggesting other mechanisms apart from its tyrosine kinase activity retained Bcr-Abl protein in the cytoplasm of CML cells. Next the subcellular distribution of Bcr-Abl was investigated in TKI-insensitive CML progenitors which survived 12-Day treatment of a potent TKI dasatinib (150nM). It was demonstrated that about 50% of Bcr-Abl was still retained in the cytoplasm of TKI-insensitive primary CML progenitors, although there was a significant increase in nuclear Bcr-Abl level in these survived cells compared to the no drug control (NDC). It was hypothesised that the cytoplasmic retention of Bcr-Abl was caused by its cytoplasmic binding partners, and it was found that a proportion of Bcr-Abl protein was associated with 14-3-3 proteins in the surviving cells, indicating the cytoplasmic retention of Bcr-Abl might be due to its binding with 14-3-3 proteins.
In addition, due to the rapid nature of kinase inhibition and nuclear transportation, studies are required to measure the earliest time-point of inhibition of Bcr-Abl tyrosine kinase by IM. The common method to do this is to employ p-CrkL which has been widely used as a surrogate marker of Bcr-Abl tyrosine kinase activity, but the accuracy of p-CrkL as an indicator of Bcr-Abl tyrosine kinase status at early time-points during in vitro TKI treatment has not been examined. It was demonstrated that p-CrkL was not a reliable indicator of Bcr-Abl kinase activity within 24 hours of IM treatment in vitro and indicated that the early responses to IM and dasatinib were different. It was also observed that there was a rapid and active dephosphorylation of Bcr-Abl within 1 hour of TKI treatment, driven at least in part by protein tyrosine phosphatase activity.
Furthermore, a farnesyltransferase inhibitor (FTI) BMS-214662 preferentially induces apoptosis in CML stem and progenitor cells compared to their normal counterparts, but another similar FTI BMS-225975 does not. However, the mechanism of action of BMS-214662 in inducing apoptosis of CML cells is not clear. When BMS-214662 was used as a negative control in the p-CrkL study, it was unexpectedly observed that the drug accumulated Bcr-Abl protein in CML cells. Thus, it was hypothesised that BMS-214662 may function as a proteasome inhibitor, which could result in accumulation of Bcr-Abl protein and further elevation of intracellular ROS level, leading to apoptosis of CML cells. Although BMS-214662 treatment induced accumulation of total ubiquitinated proteins and specifically Bcr-Abl protein in K562 cells, BMS-225975 had very similar effects, and this might result from the common mechanism of BMS-214662 and BMS-225975, which is their FTI activity. On the other hand, BMS-214662 induced significantly higher levels of intracellular ROS in both proliferating and non-proliferating K562 cells with respect to BMS-225975, indicating the production of ROS may be involved in the non-FTI mechanism of action of BMS-214662. However it was concluded that BMS-214662 was not a proteasome inhibitor like bortezomib.
Finally, the use of synthetic low density lipoprotein (sLDL) as a vehicle for drug delivery to overcome the insufficient intracellular drug concentration in CML stem cells was investigated. Uptake and internalization of unloaded sLDL particles by CML cell line K562 and for the first time by CML stem cells was observed, demonstrating the targeting potential of sLDL particles in CML when they are loaded with drugs.
Overall, this study provides further understanding of CML treatment and identifies some alternative strategies to target CML stem cells that may be used in combination with TKI to enhance the eradication of this stem cell-driven disease.
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