The Antihelmintic Flubendazole Inhibits Microtubules through a Mechanism Distinct From Vinca Alkaloids and Displays Preclinical Activity in Leukemia and Myeloma.

Abstract 1017

Poster Board I-39

On patent and off-patent drugs with previously unrecognized anti-cancer activity, could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we compiled and screened a chemical library for potential anti-leukemia agents. From these screens, we identified the antihelmintic flubendazole that is currently used for the treatment of gastrointestinal and systemic parasites, but has not been evaluated for the treatment of malignancy. To explore its efficacy as an anti-cancer agent, leukemia and myeloma cell lines were treated with increasing concentrations of flubendazole. Seventy-two hours after incubation, cell viability was measured by the MTS assay. Flubendazole reduced cell viability with an LD50 ≤ 1 μM in 8/8 myeloma and 4/6 leukemia cell lines, a concentration that is pharmacologically achievable. Likewise, flubendazole reduced the clonogenic growth of primary AML samples at nanomolar concentrations. Cell death was confirmed by Trypan blue staining. Given the effects in leukemia and myeloma cells lines, we evaluated the effects of flubendazole in mouse models of leukemia and myeloma. Sublethally irradiated SCID mice were injected subcutaneously with OCI-AML2 leukemia or OPM2 myeloma cells. Mice were then treated intraperitoneally with flubendazole (20-50 mg/kg/day – doses more than 10-fold lower than the LD50) or buffer alone. Flubendazole decreased tumor weight and volume in both mouse models up to 5-fold compared to control without evidence of weight loss or gross organ toxicity. Mechanistically, flubendazole inhibited bovine-tubulin polymerization in cell-free assays and disrupted microtubule architecture in intact cells as visualized by confocal microscopy. We demonstrated that flubendazole bound tubulin at the colchicine binding site, a region distinct from where vinca-alkaloids bind. Flubendazole arrested cells in the G2 phase of the cell cycle and increased the number of multi-nucleated cells. We also demonstrated that cell death after flubendazole treatment was related to its ability to inhibit microtubule polymerization by using cell lines with tubulin mutations (gifts from Dr F. Loganzo, Wyeth, Pearl River, NY and Drs. S. Band Horwitz and C. Yang, Albert Einstein College of Medicine, Bronx, NY). Vinca-alkaloids are p-glycoprotein (Pgp) substrates and Pgp over-expression can limit the efficacy of these agents. Therefore, we tested the effects of Pgp over-expression on flubendazole's cytotoxicty. CEM-VBL cells over-expressing Pgp remained fully sensitive to flubendazole, but were over 1000-fold more resistant to vinblastine than wild type CEM cells. Therefore, flubendazole can overcome some forms of vinca-alkaloid resistance. Given that flubendazole binds tubulin at a site distinct from vinca-alkaloids, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. OCI-AML2 leukemia cells were treated with increasing concentrations of flubendazole and vinblastine. Flubendazole and vinblastine synergistically induced cell death with combination index (CI) values of 0.09, 0.017, 0.003 and 0.001 at the EC 50, 25, 10 and 5, respectively, where CI values <1 are considered synergistic. In contrast, cell death produced by the combination of flubendazole and colchicine was closer to additive with CI values of 0.54, 0.70, 0.897 and 1.07 at EC 50, 25, 10 and 5, respectively. As the combination of flubendazole and vinblastine were synergistic in cell culture, we evaluated the combination in vivo. Mice injected subcutaneously with OCI-AML2 cells were treated with flubendazole (15 mg/kg), vinblastine (0.3 mg/kg) or the combination of the two agents. The combination decreased tumor weight and volume greater than either agent alone without behavioural changes, weight loss, or gross organ toxicity. Similar effects were observed with the in vivo combination of flubendazole and vincristine. Since neurotoxicity is a dose limiting toxicity of vinca-alkaloids such as vincristine, we evaluated the neurotoxicity of flubendazole in mice using the Tail Flick assay. At doses up to 200mg/kg daily of flubendazole, no neurotoxicity was observed. Thus, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma. Given its prior safety record when evaluated for the treatment of parasitic disease, flubendazole could be rapidly repurposed for the treatment of hematologic malignancies.