title: Investigating Intrinsic and Extrinsic Mechanisms of Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukaemia
Creators: name: Clapper, Erin M
Publisher: name: Griffith University
Year: 2021
Subjects: subject: Chronic myeloid leukaemia
subject: thioredoxin (Trx) system
subject: thioredoxin reductase (TrxR)

description: myeloid leukaemia (CML) is a myeloproliferative disorder that is responsible for 15% of all adult leukaemia cases. While the initial stages of CML are relatively mild, the terminal stage of disease, known as blast crisis, has an average survival time of approximately 12 months. CML is caused by a reciprocal chromosomal translocation that results in the production of a constitutively active non-receptor tyrosine kinase, known as bcr-abl. Bcr-abl activates a wide variety of cell proliferation and survival pathways, and this leads to abnormal cell growth and therefore cancer. Due to the involvement of bcr-abl in the progression of CML, most of the treatments for this cancer are bcr-abl specific tyrosine kinase inhibitors (TKIs). Whilst initially effective, various studies have found that resistance to TKIs occurs in 20-50% of CML cases. This is primarily linked to the highly mutagenic nature of bcr-abl. The current strategy to overcome acquired TKI resistance is to prescribe a newer generation of TKI, which is often either partly or completely ineffective. Therefore, to more effectively overcome acquired drug resistance in CML, bcr-abl independent targets need to be identified and investigated. This thesis outlines three distinct cellular systems and assesses their effect on drug resistance in CML, and their potential as targets for future CML treatments. The first system that was investigated in this thesis was the thioredoxin (Trx) system, which is involved in maintaining redox homeostasis within the cell. Upregulation of the Trx system has been associated with increased progression and poor prognosis in other cancers. In this thesis it was observed that Trx1 expression was increased in both CML cell lines and CML patient samples, compared to non-cancerous controls. Furthermore, it was found that Trx1 expression was increased in CML cells that were resistant to imatinib treatment, compared to imatinib sensitive cells. Additionally, an enzymatic inhibitor of Trx1, known as thioredoxin interacting protein (TXNIP) was downregulated in CML cells compared to non-cancerous cells, and was also downregulated in imatinib resistant CML cells. It was found that inhibiting a key element of the Trx system, thioredoxin reductase (TrxR), using chemical and specific siRNA inhibitors resulted in a decrease in the activity and expression of bcr-abl. TrxR inhibition also resulted in decreased protein expression of MYC, which is reported to regulate the transcription of bcr-abl, suggesting that downregulation of MYC could be the mechanism by which TrxR inhibitors decreased bcr-abl expression. Moreover, this thesis found that TrxR inhibition by chemical inhibitors effectively overcame imatinib induced drug resistance, further demonstrating the promising anti-cancer ability of these compounds. Inversely, the inhibition of bcr-abl by four distinct TKIs as well as bcr-abl specific siRNA resulted in decreased expression of both Trx1 and TrxR1, and decreased TrxR activity in CML cells. The inhibition of bcr-abl by TKIs is not the direct cause of apoptosis in CML, it is instead the downregulation of the downstream targets of bcr-abl; this led to the hypothesis that the inhibition of the Trx system is partly responsible for TKI induced apoptosis. Another mechanism of TKI resistance in CML that was investigated in this thesis was hypoxia, which is defined physiologically to be oxygen levels below 3%. An example of a hypoxic environment within the body is the bone marrow, where CML cells spend a large portion of their lifespan. When cells enter hypoxia, their biology changes in a multitude of ways and this has been linked to drug resistance in many cancers, including CML. The hypoxia inducible factor 1 (HIF-1) pathway is almost always linked to hypoxia-induced drug resistance. The HIF-1 pathway is only active in hypoxia and upregulates the expression of many downstream targets. In this study it was observed that CML cell growth in hypoxia was significantly decreased compared to normoxia, while cell viability and apoptosis levels remained unchanged. Furthermore, the expression of several cyclins was decreased in hypoxia, and this is likely why cell proliferation was slowed. MTT proliferation assays used to assess cell proliferation demonstrated that TKIs were far less effective in hypoxia compared to normoxia. The Trx system was also observed to be downregulated in hypoxia. However, when cells underwent reoxygenation (incubated in hypoxia and then returned to normoxia for a short period), it was found that expression of the Trx system was significantly increased, which was due to the increase in reactive oxygen species (ROS) levels. Finally, TrxR inhibitors were shown to retain most of their efficacy under low oxygen conditions, in contrast to TKIs which were ineffective in hypoxia. The final aspect of hypoxia investigated was the decreased bcr-abl protein expression observed in hypoxia, which was shown to occur via the activity of the HIF-1 pathway. Using RNA immunoprecipitation, it was found this was specifically through the downregulation of the ribosomal protein RPS6 in hypoxia, as RPS6 mediates the translation of bcr-abl. The impact of ATP binding cassette (ABC) transporters on TKI resistance in CML was also assessed. ABC transporters induce drug resistance by exporting compounds out of the cell before they can have their full effect. The upregulation of ABC transporters has been associated with increased drug resistance in various cancers. An aim of this thesis was to identify an ABC transporter that had not previously been associated with drug resistance in CML. Using publicly available RNAseq data, it was found that multidrug resistance protein 4 (MRP4) was upregulated in CML patients that were unresponsive to imatinib, compared to imatinib responsive patients. Further studies showed that MRP4 was upregulated in CML cell lines compared to non-cancerous controls, as well as in CML patients in the blast crisis phase of the disease compared to healthy donors. The effect of MRP4 activity on TKI efficacy was then assessed by specifically inhibiting MRP4 and examining differences in cell growth induced by TKIs. Using MTT proliferation and apoptosis assays, it was found that MRP4 inhibition increased the efficacy of both ponatinib and dasatinib, suggesting that MRP4 may be involved in the export of these TKIs out of the cell. Since MRP4 is reportedly under transcriptional control of Nrf2 (which also regulates expression of the Trx system), this thesis also aimed to examine any link between the Nrf2/Trx system and MRP4. Activation of Nrf2 resulted in increased MRP4 expression and activity, however, TrxR inhibitors also induced a similar response. This is because inhibiting the Trx system results in an increase in ROS, which therefore induces the activation of Nrf2. Inhibition of MRP4 also increased the efficacy of TrxR inhibitors. Inhibition of MRP4 may therefore be a promising co-treatment for current or future CML chemotherapeutics. It was shown in this thesis that MRP4 expression was upregulated in hypoxia in a HIF-1 dependant manner. Upon further investigation, it was found that this upregulation was potentially mediated by the increased expression of adenylyl cyclase 6 in hypoxia, as cAMP accumulation is a major regulator of MRP4 expression. Reoxygenation of CML cells resulted in an increase of MRP4 expression, and this was thought to be due to the increase in Nrf2 expression observed in these conditions. These two results show that changes in the oxygenation state of the cell influence the expression of MRP4 and could potentially lead to increased drug resistance. This was further investigated by inhibiting MRP4 in hypoxia and examining the effect of this on TKI efficacy. MRP4 inhibition sensitised CML cells in hypoxia to both ponatinib and dasatinib. This result reiterates the potential of using MRP4 inhibitors as a co-treatment with other CML chemotherapeutics. Overall, this thesis outlined the effect of the Trx system, hypoxia and MRP4 on TKI resistance in CML, as well as the interactions between these systems and with bcr-abl. Due to the prevalence of bcr-abl dependant forms of drug resistance in CML, alternative treatments need to be investigated and utilised. It was found that the inhibition of the Trx system using TrxR specific chemical inhibitors was able to overcome both acquired TKI resistance and hypoxia induced drug resistance. Furthermore, specifically inhibiting MRP4 activity increased the efficacy of clinically used TKIs, as well as TrxR inhibitors. MRP4 inhibition was also able to sensitise CML cells to TKIs in hypoxia. These results demonstrate that MRP4 inhibitors may also make promising co-treatments for the management of CML.