Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/92213
Type: Thesis
Title: Assessment of critical survival mechanisms exploited by BCR-ABL1+ cells to evade tyrosine kinase inhibitor-induced death: determination of novel therapeutic targets in chronic myeloid leukaemia.
Author: Schafranek, Lisa Rhiannon
Issue Date: 2014
School/Discipline: School of Medicine
Abstract: Chronic myeloid leukaemia (CML) is a clonal myeloid proliferative disease that results from constitutive activation of the Bcr-Abl oncoprotein, which disrupts normal cellular signalling potentiating the survival and maintenance of BCR-ABL1+ cells. Tyrosine kinase inhibitors (TKIs), like imatinib, have revolutionised the treatment of CML and have become the model for therapy in other cancers. Imatinib treatment also founded the paradigm that potent and continuous dosing is required for optimal patient response in patients with CML. In contrast to imatinib, the second generation TKI dasatinib has a short half-life of only 3-5 h, nevertheless a once daily dosing regime is sufficient to achieve equivalent responses to twice daily dosing suggesting that continuous and complete inhibition of Bcr-Abl may not be required for optimal response to TKI therapy. Despite initial studies indicating that a very brief exposure to a potent dose of TKI is sufficient to induce cell death in BCR-ABL1+ cells, recent studies have attributed this to sustained low-level inhibition of Bcr-Abl signalling due to inadequate drug washout. As reported in this thesis, experiments with low dose dasatinib treatment, which does not completely inhibit Bcr-Abl phosphorylation but is sufficient to induce cell death, demonstrated inactivation of STAT5 as a sensitive measure of Bcr-Abl activity. Here, it was also confirmed that <1 h exposure to potent TKI with adequate drug washout is insufficient to commit BCR-ABL1+ cells to death and it is established for the first time that at least 2 h of Bcr-Abl kinase inhibition are required. Furthermore, combinations of efficient TKI washout with specific inhibitors of STAT5, JAK and ERK ascertained sustained inhibition of pSTAT5, potentially independent of JAK2, as the determinant of commitment to cell death. Together, this research established that continuous, complete inhibition of Bcr-Abl is not required to induce cell death, but that continuous blockade of STAT5, indicative of low-level threshold Bcr-Abl inhibition, is essential, thus challenging the imatinib paradigm. Although most CML patients respond well to imatinib, only 40% of patients achieve a complete molecular response and some patients develop resistance. Blockade of Bcr-Abl signalling can drive cells to develop new survival mechanism, and amongst others, autophagy and the acquisition of extrinsic survival signalling have been implicated in resistance to therapy and/or persistent disease. Studies presented in this thesis define a role for the activation of autophagy in response to tyrosine kinase inhibition of Bcr-Abl. Induction of autophagy by TKI was confirmed using established markers of autophagy, such as the conversion of LC3-I to LC3-II, degradation of p62 and cellular morphology. Blockade of anti-apoptotic proteins Bcl-2 and Bcl-xL along with activation of stress response pathways were revealed as potential mechanisms of autophagy induction, however, further investigation into these pathways is required. Importantly, the data presented here also established clarithromycin as a novel inhibitor of TKI-induced autophagy, advocating combination treatment with TKI therapy in resistant patients. Recent observations that overexpression of cytokines and their receptors may contribute to BCR-ABL1+ cell persistence in CML patients undergoing TKI therapy. Here, the expression of IL-3 and GM-CSF cytokine receptors in BCR-ABL1+ cell lines and chronic phase CML CD34+ progenitor cells was established and signalling through those was confirmed to maintain STAT5 survival signalling, thereby protecting cells from TKIinduced death. Inhibition of JAK2 with ruxolitinib inhibited cytokine-dependent, but not Bcr-Abl-dependent, activation of STAT5 and neutralised cytokine-induced protection from cell death while having little effect in the absence of cytokines. Together, the findings of this thesis established the critical mechanisms in Bcr-Abldependent and -independent signalling that may also be targeted in combination therapeutic approaches and provides an in-depth understanding of the potential clinical effectiveness of dose reductions during dasatinib therapy. These studies will have broad implications for the ongoing development of therapeutic strategies in CML, particularly in the setting of TKI-resistance, and will aid the goal of achieving a curative treatment for patients with CML.
Advisor: Hughes, Timothy Peter
White, Deborah Lee
Melo, Junia V.
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2014
Keywords: Chronic Myeloid Leukaemia; BCR-ABL; Tyrosine Kinase Inhibitor; Dasatinib; Resistance; Autophagy; STAT5; JAK; Cytokine
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals
Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.
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