Multiple Molecular Mechanisms Contribute Towards In Vitro Resistance to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukaemia
Year: 2019
Type: dissertation
Abstract: Resistance to therapeutic drugs is detrimental to treatment efficacy in chronic myeloid leukaemia (CML). CML is driven by the constitutive activity of the tyrosine kinase, Bcr-Abl, and has been treated effectively, in the majority of patients, using tyrosine kinase inhibitor (TKI) therapy. However, therapeutic TKI resistance often results in suboptimal treatment response. The molecular mechanisms of TKI resistance include: overexpression of Bcr-Abl; perturbed activity of cell membrane influx/efflux transporters; amino acid substitutions in the Bcr-Abl kinase domain precluding TKI binding (known as kinase domain mutations); and loss of dependence on Bcr-Abl kinase activity, via the gain of alternate driver function (known as Bcr- Abl independence). There is a temporal order to the acquisition of resistance mechanisms, with shifts in clonal architecture over time and treatment altering therapy sensitivity. This thesis explores the emergence of resistance to the TKIs dasatinib and ponatinib with regard to CML, investigating the molecular mechanisms of TKI resistance, to identify novel therapeutic strategies for circumventing resistance and improving treatment outcomes. The initial experiments described development of a leukaemic cell model of TKI resistance. Using gradually increasing concentrations of the TKI dasatinib, TKI resistance was induced. A dasatinib resistant K562 cell line was established by culture in increasing concentrations of dasatinib over several months. Intermediate cell line samples were harvested over the course of TKI dose escalation and were interrogated for TKI sensitivity by TKI-induced cell death assay and phospho-CrkL IC50. Results indicated the loss of dasatinib and ponatinib sensitivity and efficacy with prolonged dasatinib exposure. The expression of BCR-ABL1 gene transcript and protein levels were analysed, demonstrating BCR-ABL1 overexpression as an early occurring resistance mechanism. The Bcr-Abl kinase domain mutation, T315I, was observed emerging late in dasatinib dose escalation, conferring complete dasatinib resistance. Dose escalation intermediates were further examined by transcriptome sequencing. By interrogating global gene expression perturbation and genetic structural rearrangements, putative resistance mechanisms were identified: a) overexpression of the drug transporter ABCG2, and b) expression of the rare BCR-ABL1 transcript isoform, e6a2. The identification of ABCG2 expression in mRNAseq experiments was validated by assays to demonstrate ABCG2 function and involvement in TKI resistance, confirming ABCG2 overexpression as a critical early resistance mechanism. Cell death and IC50 assays were performed in the presence of the ABCG2 inhibitor, Ko143, which sensitised ABCG2 overexpressing cells to TKI-based inhibition. It was demonstrated, for the first time, that ABCG2 is able to confer decreased ponatinib sensitivity. Interestingly, the loss of ABCG2 expression coincided with the gain of T315I, demonstrating the competitive advantage of T315I-positive cells. The role of the e6a2 BCR-ABL1 fusion transcript in TKI resistance was investigated. BCR-ABL1 e6a2 gene expression was quantified in dasatinib resistant cells using RT-qPCR and DNA qPCR, demonstrating a transient peak in expression, followed by a distinct reduction. Intriguingly, this reduction in BCR-ABL1 expression coincided with the gain of ABCG2. To determine whether e6a2 was less sensitive to TKI than the typical e14a2 (p210) or e1a2 (p190) Bcr-Abl fusions, these were cloned into a Ba/F3 pro-B cell line. All isoforms were able to transform cells to IL-3 independence, demonstrating the leukaemia driving activity of Bcr-Abl. TKIinduced cell death experiments determined that e6a2 expressing cells were less sensitive to dasatinib than Ba/F3 cells harbouring the e14a2 isoform. Overall, several resistance mechanisms were identified and explored, however more may remain undetected. Results suggest a model of selective pressure in drug resistance, whereby leukaemic cells expressing the most efficient drug resistance mechanisms are selected for, eventually becoming the predominant cell population. These data and conclusions have bearing on the treatment of Bcr-Abl driven leukaemia, guiding the development of better therapeutic targets and strategies.
Author: Benjamin Chia Sing Leow
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2980383927