Abstract Background: BAL27862 is a synthetic small molecule potently inducing apoptosis in cancer cells due to tubulin depolymerization. BAL27862 has a broad in vitro anti-proliferative activity against a range of human tumor lines (low nM IC50s) and elicits significant antitumor responses in animal models of human cancer, including multidrug-resistant tumors, after oral or intravenous administration. Materials and Methods: Anti-proliferative activity was analyzed with monolayer (crystal violet) or soft agar (clonogenic) assays. Effects on microtubules (MTs) were assessed by immunofluorescence (IF) or immunoblotting (IB) for -tubulin; centrosome maturation by IF for -tubulin. Results: BAL27862 showed potent anti-proliferative activity in vitro on 8 NSCLC cell lines (IC50 range 8.5–17nM). Significant activity was also observed on 3 patient-derived NSCLC lines, highly resistant to paclitaxel (clonogenic IC50s: paclitaxel >3.5 M; BAL27862 15nM, 29nM, 300nM). In all tumor histotypes analyzed, BAL27862 elicited a unique MT phenotype, distinct from that observed with conventional MT-targeting agents (paclitaxel, vinblastine, colchicine) or other mitotic inhibitors. In proliferating A549 NSCLC cells (anti-proliferative IC50: 16±2nM; MT depolymerization IC50: 10–20nM) BAL27862 caused a concentration-dependent effect on MTs. In interphase cells, at concentrations ≥15nM the MT network collapsed towards the MT-organizing center, with an absence of peripheral MTs. In dividing cells, an apparent fragmentation of the mitotic spindle occurred at 15nM, associated with the appearance of tiny MT asters. The asters decreased in size as concentrations increased (15–100nM), and were co-localized with condensed chromatin. A comparative titration (5–100nM) with the clinically used MT destabilizer vinblastine (VBL) indicated no phenotypic overlap. VBL-treated interphase cells had a mesh-like MT network with peripheral MTs intact, while in dividing cells aberrant, spindle-like structures were observed at concentrations ≥15nM. Using non-immortalized HS68 human fibroblasts, no obvious effects of BAL27862 (10–20nM) on centrosome number or size were observed, as defined by -tubulin staining; in contrast to an increase in size and decrease in apparent resolution of the centrosomes after treatment with VBL at the same concentrations. BAL27862 treatment also converted the abnormal spindle structures induced by the MT destabilizers VBL or colchicine into the distinct and dominant BAL27862 MT phenotype, further highlighting it's novel mechanism of MT disruption. Conclusions: BAL27862 is a new tubulin-interacting agent with a novel mechanism of action leading to a unique MT phenotype. These observations, together with a broad antitumor activity targeting refractory tumors, strongly support further development of BAL27862 as an anticancer agent. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C229.