Abstract 3789: BAL101553 (prodrug of BAL27862): the spindle assembly checkpoint is required for anticancer activity

Abstract

Abstract Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule (MT)-depolymerizing agent with a broad in vitro anti-proliferative activity against human tumor lines refractory to standard MT-targeting agents. The prodrug has anti-cancer activity in diverse tumor models when given orally or iv; alone or in combination therapy. In this study, the role of the ‘Spindle Assembly Checkpoint’ (SAC) in the mechanism of action of BAL27862 was evaluated. Methods: To evaluate BAL27862-resistance mechanisms, tumor lines were selected in vitro by increasing drug levels. Anti-proliferative activity was analyzed using Crystal Violet assay. siRNA transfection for 24 h was followed by drug treatment (50 nM) for 48 h. Mitotic index was evaluated after 18 h treatment by p-Histone H3 staining (pHH) or flow cytometry (FC). SAC complex formation was assessed by BubR1 immunoprecipitation (IP) and immunoblotting for SAC components. Results: The SAC ensures cell cycle arrest upon spindle disruption, promoting tumor cell death after prolonged mitotic arrest. SAC formation involves phosphorylation and assembly of a multiprotein complex, of which BubR1 and Mad2 are essential components. Using siRNA approaches, BubR1 was shown to be required for BAL27862 anti-proliferative activity in 5 BAL27862 sensitive tumor lines (IC50s: 8-20 nM). A similar analysis of Mad2 in 3 lines resulted in almost identical attenuation of the effect of BAL27862. For example, non-targeting control siRNA (NTC)-treated H460 lung cancer cells entered a cell death program following BAL27862 treatment (∼45% cell death vs. starting cell number); a response attenuated by BubR1 and Mad2 down regulation (71% and 62% proliferation resp. vs. DMSO controls). The accumulation of mitotic arrested (pHH positive) cells associated with BAL27862 treatment was also absent after BubR1 or Mad2 siRNA transfection. To investigate whether BAL27862 (50 nM, 18 h) induces the formation of the SAC, BubR1 IPs from 3 sensitive tumor lines (IC50: HeLa cervical: 20 nM; A549 lung: 15 nM; SKOV3 ovarian: 12 nM) were analyzed for SAC components. In all cases, complex formation was observed, consistent with a concomitant mitotic accumulation as measured by FC. Strikingly, in variants of the same lines selected for BAL27862 resistance (resistance factors: 3.2-9.4) no SAC formation or mitotic accumulation was observed under the same conditions. Moreover, BAL27862-induced protein mobility shifts, consistent with activating phosphorylation of SAC components, were also absent in the resistant variants. Conclusions: The association of SAC activation with BAL101553sensitivity suggests prediction of cancer response may lie in its tumor checkpoint control function. BAL101553 is currently in Phase 2a clinical evaluation in advanced cancer patients. Exploratory biomarkers are being evaluated to aid selection of patients most likely to respond. Citation Format: Felix Bachmann, Karin Burger, Heidi Lane. BAL101553 (prodrug of BAL27862): the spindle assembly checkpoint is required for anticancer activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2015-3789