INCREASED THERAPEUTIC INDEX OF TYROSINE KINASE INHIBITORS IN CML THROUGH MEMBRANE TRANSPORTER UPTAKE

Abstract

Chronic myeloid leukaemia (CML) is a hematopoietic stem cell disease, distinguished by the BCR-ABL gene and the presence of the Philadelphia chromosome. Recent studies have revealed that CML resistance is due to up regulation of the membrane and drug transporters ABCB1 and ABCG2, which have a critical role in drug resistance. Indeed, there is strong evidence suggesting drug transport occurs mainly via proteinaceous carriers. In this regards, the MayBridge Rule of 3 Fragment Library (May Ro3) is a relatively small collection of chemical moieties that are pharmacophore rich and ‘metabolite-like’. As a result, in the current project we demonstrate the application of 'binary weapons' which are small non-toxic molecular agents with high affinity to both transporters and imatinib. The interaction between the binary weapon and imatinib enhances the anti-cancer impact of imatinib. In this research study, the first 500 fragments from the Maybridge Fragment (MBF) library was screened to find suitable candidates for enhancing the imatinib effect on K562 cell line while the MBFs alone possessed no toxic effect. Firstly the screenings were performed by pooling fragments, in which we tested the K562 cell enhanced toxicity of imatinib plus MBFs via MTT assays and then those selected pools were analysed in single MBF screenings. Then titration assays of those selected single hits were performed to find the most effective concentration for selected hits. Finally, the selected single hits were used in qPCR to check the effect on expression of candidate membrane associated transporters that were identified from literature review. Moreover, in all experiments SH-SY5Y cells and the drug cisplatin were employed as controls for K562 and imatinib. In pooled screens for K562 and imatinib, 40 fragments were selected and these pools were further investigated using single MBF screenings and 6 of the 40 MBFs showed the desired effect. Following titration experiments, 2 MBFs were shown to carry the least toxicity even in the highest concentrations, so they were selected as candidates for investigation of the impact on the expression of transporters genes. The expression of transporter mRNAs was investigated and showed some significant dys-regulation of influx or efflux transporters. Compared to the control group, when K562 cells were treated with MBFs (161 or 247), all transporters, efflux and influx, showed downregulation. However, treatment with imatinib and MBF No. 161 for 1 hour showed downregulation for ABCB10, BCR-ABL and SLCO1A2SLCO1A2(P-value = 0.001). However, the expression for other transporters were not dysregulated (no upregulation no downregulation) (P value=0.001), showing the same expression with the control group. Moreover, treatment of K562 cells with imatinib and MBF hit No. 247, all influx and efflux transporters were downregulated significantly. Lastly, the BCR-ABL expression in all different conditions showed downregulation. In this research programme, K562 served as target cells and as a model of CML while SH-SHY5Y were employed as non-target cells. Through using the 2 selected candidate MBFs in conjunction with imatinib, the cytotoxicity of imatinib was shown to prevail within K562 cells and not SH-SHY5Y cells; in essence increment of the therapeutic index of imatinib was demonstrated.

Date of Award	31 Dec 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Douglas Kell (Supervisor), Philip Day (Supervisor) & Malkhey Verma (Supervisor)