Abstract 3795: BubR1 function is required for the antiproliferative activity of the novel microtubule-targeting drug BAL27862 (active moiety of the prodrug BAL101553)

Abstract

Abstract Background: BAL27862 is a novel small molecule that induces apoptosis in cancer cells through microtubule destabilization associated with a distinct microtubule phenotype. BAL27862 has broad antitumor activity in preclinical models of human cancer, and has recently entered Phase I clinical trials in advanced cancer patients administered as a highly soluble lysine prodrug (BAL101553). Methods: Combinatorial RNAi screening using HeLa cervical carcinoma cells and a siRNA library targeting the human kinome/phosphatome was followed by image analysis of cell number and fraction with normal morphology after optimal BAL27862 treatment (50nM, 24h). BubR1 siRNA effects were also assessed by immunoblotting (IB) and 48h crystal violet proliferation assays. Drug-resistant tumor lines were selected in vitro by incubation with increasing BAL27862 concentrations. Patient-derived tumors were maintained in mice and analyzed by immunohistochemistry (IHC). Results: Combinatorial RNAi screening identified BubR1 (BUB1B gene) as a protein required for the antiproliferative and phenotypic effect of BAL27862. This finding was confirmed using single independent siRNAs. BubR1, an evolutionary conserved protein kinase controlling cell division, is a core component of the spindle assembly checkpoint. Consistent with this, a more detailed siRNA analysis across a panel of lines derived from different histological backgrounds (breast, colon, lung, brain) confirmed the essential role of BubR1 in the in vitro response of diverse tumor lines to BAL27862 treatment. For example, non-targeting control siRNA (NTC)-treated MDA-MB-453 breast carcinoma and H460 NSCLC cells entered a cell death program following BAL27862 treatment (∼60 % & ∼40 % cell death resp. vs. starting cell number); a response clearly attenuated by BubR1 down regulation (∼80% cell proliferation. vs. DMSO controls). Further IB analysis of tumor cell variants selected for stable drug resistance to BAL27862 (∼3-24-fold resistant), indicated that BubR1 protein expression was down regulated in 3 of 5 variants as compared to parental lines (SKOV3 ovarian, A549 & H460 non-small cell lung cancer [NSCLC]). Hence, reduced BubR1 expression may modulate development of resistance to BAL27862 under treatment. Strikingly, IHC analysis of paired patient-derived tumors derived from gastric and NSCLC carcinomas, demonstrated that intrinsic resistance to BAL27862 treatment ex vivo can also be associated with reduced BubR1 tumor expression. Conclusions: Reduced BubR1 expression correlates with resistance to BAL27862 treatment in a number of experimental models. Further work to understand the functional significance of BubR1 to the mechanism of action of BAL27862 is ongoing to explore the potential of using this biomarker to support patient stratification efforts during the clinical development of BAL101553. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3795. doi:1538-7445.AM2012-3795