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Type: Theses
Title: In vitro investigation of intracellular ponatinib transport and modelling ponatinib resistance in BCR-ABL1+cell lines: implications for therapeutic strategies
Author: Lu, Liu
Issue Date: 2016
School/Discipline: School of Medicine
Abstract: The use of tyrosine kinase inhibitors (TKIs), which target Bcr-Abl, has become the first-line treatment for chronic myeloid leukemia (CML). However, TKI resistance remains a major impediment to successful treatment of CML. The novel third generation pan-Bcr-Abl TKI ponatinib has demonstrated efficacy in overcoming single BCR-ABL1 kinase domain (KD) mutation based resistance including BCR-ABL1T315I [T315l superscript], which inhibits the binding of all other available TKIs. While intracellular transport of the first and second generation TKIs have been studied, little is known about the complex interactions between ponatinib and drug transporters. Additionally, clinically significant mechanisms that may result in resistance to ponatinib remain to be elucidated. In this study, we investigated the interaction of ponatinib with drug transporters, and emerging modes of ponatinib resistance in previously TKI-naive and dasatinib resistant BCR-ABL1+ cell lines. This study examined the role of ABCB1, ABCG2 and OCT transporters in ponatinib efflux and influx, as these transporters have been previously implicated in the transport of other TKIs. Results demonstrated neither ABCB1 ABCG2 nor OCT-1, play major roles in ponatinib transport. In addition, data revealed that ponatinib transport is not ATP/temperature dependent, and therefore is most likely to be passive. To investigate potential resistance mechanism(s), ponatinib resistance was generated by exposure to increasing concentrations of ponatinib in BCR-ABL1+ cell-lines that either priorly treated with a TKI (dasatinib) or naive to all TKIs. Two resistant cell lines, previously resistant to dasatinib and then treated with ponatinib, demonstrated the emergence of BCR-ABL1 KD mutation(s). In one of these cell lines, the level of T315I increased from 44% to 66%, with BCR-ABL1 mRNA expression also increasing. In the second cell line the compound mutation G250E/E255K developed. In contrast, the TKI (imatinib, nilotinib and dasatinib) naive ponatinib resistant cell lines did not demonstrate BCR-ABL1 KD mutations. Conversely, both of these resistant lines developed Bcr-Abl-independent resistance via Axl overexpression. Axl, a receptor tyrosine kinase, has previously been associated with TKI resistance. My studies are the first to report it in association with ponatinib resistance. In agreement with the observation that Axl overexpression causes ponatinib resistance, ponatinib sensitivity was restored following Axl inhibition or shRNA-mediated-knockdown of Axl. In conclusion, the studies outlined in this thesis reveal that unlike other TKIs, ponatinib is not transported by ABCB1, ABCG2 or OCT-1 and therefore patients are unlikely to be susceptible to resistance caused by deregulation of these transporters. Moreover, the study also identified that in the setting of prior TKI-exposure, Bcr-Abl dependent mechanisms, such as BCR-ABL1 KD mutations and BCR-ABL1 mRNA overexpression, are likely to cause ponatinib resistance. However, in the TKI-naive setting, Bcr-Abl-independent modes of resistance develop preferentially, and Axl presents as a key mediator of this resistance. While further studies are required, particularly in reference to Axl expression in patients being treated with ponatinib, these data may suggest that combination therapeutic approached may be the most efficacious in the setting of up=front ponatinib use and subsequent development of resistance.
Advisor: White, Deborah Lee
Hughes, Timothy Peter
Dissertation Note: Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2016.
Keywords: CML
TKI resistance
Research by Publication
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:
DOI: 10.25909/5ba46e34cb6db
Appears in Collections:Research Theses

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