Characterisation of novel anticancer agents in childhood acute lymphoblastic leukaemia

Tubulin-binding agents (TBAs) are employed widely in cancer treatment, including childhood acute lymphoblastic leukaemia (ALL). Despite their success, resistance to TBAs can be a major clinical problem. Mechanisms mediating resistance to TBAs that target the colchicine-binding site on β-tubulin, along with novel therapeutic strategies were investigated. 2-Methoxyestradiol (2ME2) is a multi-targeted TBA active in various cancer types, yet its efficacy and mechanisms(s) of action in haematological malignancies are not well evaluated. To improve understanding of mechanisms(s) of action and drug resistance, leukaemia cells, CCRF-CEM, were selected for resistance to 2ME2. These resistant cells displayed reduced drug-induced mitotic arrest and increased tubulin polymer levels. Moreover, the resistant cells also acquired β-tubulin mutations that affected microtubule stability and induced conformational change(s) to the 2ME2-binding site on β-tubulin. Two of the mutations resided in the colchicine-binding site, yet did not affect colchicine sensitivity, suggesting that 2ME2 and colchicine differ in their binding to β-tubulin. These 2ME2-resistant leukaemia cells have provided novel insights into microtubule stability and drug-target interactions to the tubulin molecule. Differential proteomics analysis identified alterations in the expression of cytoskeletal proteins in the 2ME2-resistant leukaemia cells, including a significant increase in class II β-tubulin. Furthermore, γ-enolase and galectin-1 were up-regulated and class IVb β-tubulin was down-regulated in 2ME2-resistant cells. Gene specific knockdown of class II β-tubulin expression in neuroblastoma cells using small interfering RNA increased sensitivity to 2ME2 and other colchicine-binding agents, suggesting that class II β-tubulin plays a functional role in drug resistance and sensitivity. In a clinically relevant NOD/SCID ALL mouse xenograft model, orally active 2ME2 and its analogue, ENMD-1198, increased survival rates in human ALL xenografts compared to control mice. ENMD-1198 showed better activity than 2ME2 against the ALL xenografts. Combining two microtubule-destabilising agents, ENMD-1198 and vincristine, demonstrated synergistic effect by causing microtubule disruption, angiogenesis and cell proliferation inhibition, and apoptosis induction in leukaemia cells. Importantly, ENMD-1198 and vincristine combination was more potent than single agent treatment in a vincristine-resistant ALL xenograft. In conclusion, this thesis identified novel mechanisms of resistance to 2ME2 and developed a new therapeutic strategy that could improve the treatment of drug refractory childhood ALL