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Type: Thesis
Title: TKI resistance in CML cell lines : investigating resistance pathways.
Author: Tang, Carine
Issue Date: 2012
School/Discipline: School of Medicine
Abstract: Chronic myeloid leukaemia (CML) is characterised by the presence of the Philadelphia chromosome which harbours the Bcr-Abl oncogene. BCR-ABL is a constitutively active tyrosine kinase that can be inhibited by rationally designed tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib and dasatinib. Although TKI therapy is an effective treatment for many patients, resistance can arise. There are currently four identified resistance mechanisms. These are 1) overexpression of drug-efflux proteins (ABCB1 and ABCG2), 2) BCR-ABL kinase domain (KD) mutations, 3) increased BCR-ABL expression and 4) BCR-ABL independent mechanisms such as Lyn kinase expression. In this study the interplay between these four recognised modes of TKI resistance is investigated. Imatinib- and dasatinib-resistant cell lines were established and used to investigate TKI resistance in vitro. Viability and IC50 assays were used to demonstrate TKI sensitivity/resistance. Flow cytometry was used to screen for ABCB1 and ABCG2 cell surface expression, while conventional sequencing and the MassARRAY method were used to determine the mutation status of the BCR-ABL KD. Fluorescence in situ hybridisation (FISH) and quantitative DNA PCR were used to investigate Bcr-Abl DNA copy number, and RQ-PCR was used to investigate expression levels of BCR-ABL and Lyn mRNA. These studies revealed that IM-resistant K562 cell lines exhibited increased BCR-ABL expression at the onset of resistance. Interestingly, these cell lines had increased viability and IC50s for IM and NIL, while the DAS IC50s were variable. Further investigation revealed Lyn overexpression in the cell line which was more sensitive to DAS. The development of a DAS-resistant K562 culture resulted in the emergence of the T315I mutation. Studies of the intermediate stages of resistance of this DAS- resistant cell line revealed that increased BCR-ABL expression occurred gradually, preceding the emergence of the mutation, at which time the BCR-ABL expression decreased and plateaued. Thus, it appears that increased BCR-ABL expression may be the initial mechanism of resistance, followed by the emergence of a KD mutation which has a clear selective advantage. This phenomenon was observed a further four times (in a DAS-resistant K562 Dox culture, and in three IM-resistant KU812 cultures) each time with the emergence of different KD mutations. Different KD mutations resulted in differential resistance to the three TKIs used in this study. In contrast, three IM-resistant K562 Dox cell lines were not found to have any KD mutations, nor BCR- ABL overexpression. Instead, the primary cause of resistance in these lines appeared to be an increase in ABCB1 expression. The addition of PSC833 (an ABCB1 inhibitor) decreased the IM, NIL, and DAS IC50s for all three resistant lines to the level of the naïve control. This indicated that ABCB1 expression, facilitating active efflux of the drugs, is the primary mechanism of resistance in these lines. This study demonstrates that KD emergence is a stochastic event, as the same mutation did not always occur twice when exposed to the same TKI. However, increased ABCB1 expression was more likely to arise recurrently in the predisposed K562 Dox cell line. Notably, different TKIs elicited different resistant mechanisms, and all but one (the Lyn overexpressing K562 cell line) were BCR-ABL dependent. Furthermore, all resistant cell lines showed cross-resistance (at least to some extent) to the three TKIs tested, suggesting that currently available TKIs share the same susceptibilities to drug resistance.
Advisor: Hughes, Timothy Peter
White, Deborah Lee
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2012
Keywords: CML; imatinib; nilotinib; dasatinib; TKI; resistance
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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