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Antimicrotubule agents are highly effective in the treatment of acute lymphoblastic leukemia (ALL), but resistance to these agents can be a significant clinical problem. Hence, the development of novel multi-targeted small molecules are essential to treat drug resistant disease. One such agent is 2-methoxyestradiol (2ME2) which displays anti-angiogenic and anti-tumour effects in various cancers, although studies in ALL have been limited. 2ME2 binds to β-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. The exact mechanism(s) of action of 2ME2 remain unclear and the aim of this study was to determine the pathways involved in the mechanism of action and resistance to 2ME2 in leukemia cells. To address these aims, 2ME2-resistant cells were generated by selecting T-cell ALL, CCRF-CEM, cells to step-wise increases in 2ME2. Four sublines, CEM/2ME2-3.6R, CEM/2ME2-7.2R, CEM/2ME2-14.4R and CEM/2ME2-28.8R, were chosen for detailed analysis and were found to be 11-, 47-, 88- and 107-fold resistant to 2ME2, respectively. Although 2ME2 and colchicine are believed to have overlapping binding sites, the 2ME2 resistant sublines were hypersensitive to colchicine. Surprisingly, the 2ME2 resistant sublines were hypersensitive to epothilone B, an agent that shares a common binding site with paclitaxel. Yet the resistant cells were not hypersensitive to paclitaxel. The ability of 2ME2 to cause cell cycle arrest in CEM cells was found to be concentration-dependent and at 10uM 2ME2, parental CEM cells exhibited marked G2/M arrest. In contrast, 10-fold higher concentrations of 2ME2 were required for G2/M arrest in the CEM/2ME2-28.8R cells. Increased levels of polymerized tubulin were detected in the resistant cells with CEM/2ME2-28.8R cells having 48.62 ±3.91% (P<0.05) polymer, compared to CEM cells 29.05 ± 0.94%. The increased levels of microtubule polymer corresponded with increased levels of acetylated α-tubulin. Genomic sequencing identified four distinct heterozygous βI-tubulin gene mutations in the resistant cells. The CEM/2ME2-3.6R acquired a D197N mutation whereas 2ME2-7.2R and 2ME2-14.4R cells acquired an additional mutation, S25N. Furthermore the most highly resistant 2ME2-28.8R cells acquired two extra mutations, A248T and L350N. Three dimensional structural investigations of the αβ-tubulin heterodimer revealed that the A248T and L350N encompass the colchicine binding site yet the cells harbouring these mutations are not cross resistant to colchicine. In contrast, mutations D197N and S25N are distinctively remote from the colchicine binding site, suggesting their observed effects may be indicative of an induced conformational change to the binding site. The 2ME2-resistant leukemia cells provide novel insights into microtubule stability and, in particular, drug-target interactions.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA