Background:The cytoskeleton of eukaryotic cells participates in various cellular functions such as motility, secretion, signaling and proliferation. Microtubules (MTs) are an integral part of the cytoskeleton. Among anti-cancer agents, drugs targeting tubulin or MTs are among the most, if not the most, effective class of agents. The list of compounds that bind to tubulin or the MTs is large and continues to expand. The overwhelming majority are natural products, and their chemical structures are remarkably diverse. The vinca alkaloids were introduced in the 1950's, and although they were useful in a wide range of malignancies, interest in developing new agents targeting MTs gradually declined, until the introduction of Taxol. Arguably the most effective agent since cisplatin, the remarkable activity of Taxol stimulated interest in tubulin and MTs as targets for chemotherapy. The clinical success of Taxol has led to a wealth of new scientific knowledge, reinforced the importance of the tubulin/MT system as a target for cancer chemotherapy and spurred efforts to identify novel tubulin-active agents. In keeping with the research focus of the laboratory on the problem of drug resistance, we initially began studies aimed at identifying non-Pgp mechanisms of paclitaxel resistance. Selections performed with paclitaxel in the presence of verapamil succeeded in isolating cell lines with acquired resistance to paclitaxel that did not over-express MDR 1. Characterization of these cells led to the identification of mutations in the predominant tubulin isotype, M40. Similar studies performed using two additional tubulin stabilizing agents, epothilone A and epothilone B, led to the isolation of drug resistant cells which were also shown to harbor mutations in beta tubulin. Using a refined molecular model of tubulin, and guided by the mutations identified in our drug resistant cell lines and the cross-resistance profile of these cells, we were able to dock both paclitaxel and the epothilones in the putative binding pocket and propose a common pharmacophore for the taxanes, and the epothilones. In the field of MT targeting agents (MTAs), our current research goals are to (1) to increase our understanding of how MTAs interact with tubulin and lead to cell death; (2) understand the mechanisms of resistance to MTAs; and (3) develop assays to monitor the pharmacodynamics of MTAs in patients. In the clinic, we continue to conduct trials examining MTAs.Project Description and Plans: Given our success in identifying mutations in paclitaxel- and epothilone-resistant cells, and encouraged by the information accumulated and the lessons learned, we began selections with HTI-286 a synthetic hemiasterlin in development by Wyeth-Ayerst. The hemiasterlins are sponge-derived tripeptides that depolymerize existing MTs and inhibit MT assembly. Since hemiasterlins are poor Pgp substrates, they have been considered attractive candidates for cancer therapy. We were interested in the hemiasterlins for several reasons: (1) We had worked with paclitaxel and the epothilones, two agents that promote MT polymerization, and we were interested in investigating a depolymerizing agent; and (2) Unlike paclitaxel and the epothilones, the binding site of the hemiasterlins is less well defined, and it offered us the opportunity of contributing to its elucidation by identifying crucial residues as we had done with paclitaxel and the epothilones. The basis of resistance to HTI-286 was examined in cell populations derived from A2780/1A9 cells selected in HTI-286. A2780/1A9-HTI-resistant cells (1A9-HTIR series) were 57- to 89-fold resistant to HTI-286. Cross-resistance (3 to 186 fold) was observed to other MT depolymerizing drugs, with collateral sensitivity (2 to 14 fold) to tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble tubulin in 1A9 parental and 1A9-HTIR cells corroborated the HTI-286 cytotoxicity data. Specifically, we were able to demonstrate the presence of more stable MTs in the resistant cell populations by several criteria. In addition, we were able to demonstrate post-translational modifications on a-tubulin consistent with increased MT stability: acetylation and detyrosination. Tubulin can undergo numerous post-translational modifications, including phosphorylation, polyglutamylation, polyglycylation, deglutamylation, acetylation and tyrosination/detyrosination. Although the functions of the modifications on tubulin remain unclear, deglutamylation, acetylation and detyrosination, occur on a-tubulin and are at least indicative of stable MTs. Acetylation occurs on lysine 40 of a-tubulin and does not appear to increase the levels of stable MTs but instead accumulates on existing stable MTs. Glu-tubulin is formed when the last residue on a-tubulin, a tyrosine, is removed by tubulin-carboxypeptidase and the glutamic acid is exposed. Detyrosination only occurs on a-tubulin in polymerized MTs and restoration of the tyrosine residue to a-tubulin requires tyrosine-ligase and occurs exclusively on soluble a/b-tubulin heterodimers. We observed a marked increase of both acetylated and detyrosinated a-tubulin in the resistant cells, indicating the presence of more stable MTs. The 1A9-HTIR cells exhibited either a single nucleotide change in the M40 b-tubulin isotype: S172A; or in two cell populations without mutations in b-tubulin, mutations in the Ka-1 a-tubulin isotype: S165P and R221H in one resistant cell population and I384V in another. We believe these mutations (located in areas of intra- and inter-dimer contact) enhance MT stability and confer resistance to HTI-286 and other MT depolymerizing agents while simultaneously increasing susceptibility to MT polymerizing drugs. None of the residues mutated in our resistant cells are located in the region of a-tubulin implicated in HTI-286 binding. As we were investigating the hemiasterlin resistant cell lines, we were also conducting a phase II clinical trial with BMS-247550 (ixabepilone) in patients with renal cell carcinoma. BMS-247550 is an epothilone B analogue and MT-stabilizing agent. As a part of this ongoing trial, we were attempting to obtain tumor biopsies before therapy and after the fifth dose of BMS-247550. The original goal had been to examine the pharmacodynamics of BMS-247550 by quantitating the degree of tubulin polymerization before and after drug administration. This measurement would allow us to establish whether BMS-247550 had stabilized MTs in the tumor cells. However, we also considered alternate methods to demonstrate MT stabilization. Encouraged by the results in our hemiasterlin resistant cells where tubulin acetylation and detyrosination had been correlated with MT stabilization, we chose to investigate these chemical modifications in the patient samples. As a first step we demonstrated that the levels of detyrosinated (glu-terminated) and acetylated a-tubulin correlated well with MT stabilization induced by BMS-247550 in cultured renal and ovarian cancer cells, suggesting these modifications could be used to monitor MT-stabilization. More importantly, in examining the patient samples we found that after treatment with BMS-247550, the levels of glu-terminated and/or acetylated a-tubulin increased 2- to 100-fold in 8 out of 8 serial tumor biopsies. These data indicate BMS-247550 reached the tumors and engaged the MT target, leading to MT stabilization. We conclude that glu-terminated and/or acetylated a-tubulin levels are simple and reliable markers for the pharmacodynamic effects of BMS-247550. We believe that assessing post-translationally modified tubulin levels may provide a simple and reliable assay of the pharmacodynamic effects of other MTAs. It is interesting that despite the widespread use of the MT-stabilizing taxanes, in vivo demonstration of drug-target interaction has been very limited.
Showing the most recent 10 out of 11 publications
