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Type: Thesis
Title: Evaluation of anti-proliferative and pro-apoptotic effects of tyrosine kinase inhibitors on CML-CD34+ cells.
Author: Hiwase, Devendra
Issue Date: 2010
School/Discipline: School of Medicine
Abstract: Although imatinib (IM) has revolutionalised CML management, 30 to 40% patients fail IM therapy. Many of these patients can be rescued with second generation tyrosine kinase inhibitors (TKI), dasatinib, nilotinib and bosutinib. This research elucidates the dasatinib cellular transport pathways and its role in mediating dasatinib resistance. It also assesses dynamics of Bcr-Abl kinase inhibition and apoptosis in CML lines and CML-CD34+ progenitors. Lastly it addresses the role of cytokines in mediating TKI resistance and possible combination therapy to circumvent cytokine mediated TKI resistance. The organic cation transporter (OCT-1) mediates IM influx and low OCT-1 activity is a major contributor to suboptimal response in CML patients treated with IM. In the current study the relevance of OCT-1 activity and efflux pumps in determining intracellular concentration (IUR) of dasatinib were assessed. In contrast to IM, dasatinib cellular uptake is not significantly affected by OCT-1 activity, so that expression and function of OCT-1 is unlikely to affect response to dasatinib. Dasatinib is a substrate of efflux proteins, ABCB1 and ABCG2. Overexpression of these proteins can mediate dasatinib resistance. There is increasing evidence that nilotinib is an ABCB1 inhibitor. These different interactions of dasatinib and nilotinib with ABCB1 were exploited for combination therapy. Nilotinib increased 14CDasatinib IUR and had synergistic effect in inducing cell death in ABCB1 overexpressing cells. These data suggest that combinations of these two TKI can overcome ABCB1 mediated dasatinib resistance and may allow the use of lower concentrations of each drug. In contrast to IM, dasatinib cellular influx is predominantly passive and maximum intracellular concentration is achieved within a few minutes. This was further confirmed by the observation of maximum Bcr-Abl kinase inhibition within 30 minutes of culture with dasatinib. Despite reactivation of Bcr-Abl kinase within 30 minutes of drug washout, short-term (30 minutes) intense (>90%) Bcr-Abl kinase inhibition with dasatinib triggers apoptosis in CML cell lines. This is in contrast to the previously established paradigm that continuous kinase inhibition is required for optimal response to IM. These results were further supported by a recently published dasatinib dose optimisation study. Further work in this thesis demonstrated that although Bcr-Abl kinase reactivates within 30 minutes of drug washout, the prosurvival proteins Erk, AKT and STAT5 dephosphorylated rapidly while the apoptotic proteins remained phosphorylated. This differential degradation of prosurvival and apoptotic proteins might be responsible for a state of “oncogenic-shock”, as described by Sharma et al. Subsequent studies demonstrated that in the absence of cytokines, short-term intense Bcr-Abl kinase inhibition with therapeutically achievable concentration of dasatinib (100 nM dasatinib) eliminated 70 to 80% of CML-CD34+ progenitors. However, in the presence of cytokines despite >90% Bcr-Abl kinase inhibition it did not trigger cell death in CML progenitors. These results suggest that intense Bcr-Abl kinase inhibition alone may not be adequate to trigger cell death in CML progenitors. Further studies demonstrated that cytokines mediate TKI resistance by activating JAK2-STAT5 pathway and that the combination of JAK2 inhibitor and TKI can circumvent cytokine mediated TKI resistance.
Advisor: Hughes, Timothy Peter
Kumar, Sharad
Vaz de Melo, Junia
White, Deborah
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010
Keywords: chronic myeloid leukemia; dasatinib; cytokines
Provenance: Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.
Appears in Collections:Research Theses

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