Abstract A183: The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expression-dependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo
Authors: Berges Raphael , Tchoghandjian Aurélie , Honore Stephane , Figarella-Branger Dominique , Bachmann Felix , Lane Heidi , Braguer Diane 
Published: May 2016

Abstract
Background: BAL101553 is the prodrug of the novel small molecule BAL27862, undergoing clinical evaluation in advanced cancer patients as an i.v. (phase 2a) and oral (phase 1) formulation. BAL27862 binds the colchicine site of tubulin with distinct effects on microtubule organization, resulting in activation of the ‘spindle assembly checkpoint’ and tumor cell death. BAL27862 has broad activity against diverse cancer models, including tumors refractory to conventional treatments. The drug efficiently distributes to tumor and to brain, with cytotoxic effects in glioblastoma (GBM) lines. We have shown that End-binding 1-protein (EB1) overexpression correlates with GBM progression and sensitizes GBM tumors to Vinca alkaloids. Moreover, EB1 is overexpressed in the GBM stem-like cells, GBM6, that display a high tumorigenicity with an infiltrative pattern of migration in vivo. Here, we investigate the activity of BAL27862/BAL101553 on GBM6 stem-like cells according to EB1 expression level in vitro and in orthotopically transplanted nude mice.
Material and methods: Effects of BAL27862 on wild-type and EB1-downregulated GBM6 cells were analyzed using sulforhodamin B survival, clonogenic and transwell migration assays. Nude mice were orthotopically grafted with GBM6GFPSh0 (EB1+) and GBM6GFPShEB1 (EB1-) cells. BAL101553 (25mg/kg) or vehicle were administrated i.v. at D30, D33 and D36. Stem cell phenotype characterization, tumor volume and brain invasion were analyzed at D45, D75 and D105. Overall survivals were analyzed. Stem-like cell differentiation was analyzed by flow cytometry and real-time PCR by analyzing stem cell markers (A2B5, CD133), markers of differentiation (GFAP, β-III-tubulin and CNPase) and self-renewal assay.
Results: BAL27862 inhibited GBM6 survival after 72h (half maximal effective concentration [EC50] = 20nM) with EB1-downregulation resulting in a ∼2-fold increase in EC50 (40nM). BAL27862 inhibited clonogenicity and cell migration in GBM6 cells, even at a low, non-cytotoxic concentration (6 nM). However, these effects were only detectable with cytotoxic concentrations (≥ 20 nM) in EB1 downregulated GBM6 cells. Just three BAL101553 administrations over a week provoked a mice survival increase of 69 and 32 days (vs. vehicle controls) after GBM6GFPSh0 and GBM6GFPShEB1 tumor grafting, respectively. Analysis of the pattern of invasion, and quantification of fluorescent tumor cells and A2B5+cells in brain, demonstrated that control cells were more invasive than EB1-downregulated cells, and that anti-invasive effects of BAL101553 were more potent in control cells than in EB1-downregulated cells. BAL27862 also acted on stem-like cells by inducing apoptosis and differentiation, as shown by the decrease in self renewal and stem cell markers.
Conclusion: BAL27862 is the first tubulin-binding agent to show a strong inhibitory activity against stem-like cell proliferation and invasion. These results support further investigation of BAL101553 for the treatment of GBM patients, with EB1 expression a potential predictive biomarker for drug responders.
Financial supports: (SIRIC label) INCa-DGOS-Inserm 6038, A*MIDEX project “Investissements d'Avenir” (No. ANR-11-IDEX-0001-02), the ITMO Cancer AVIESAN as part of the Cancer Plan and Basilea Pharmaceutica.
Citation Format: Raphael Berges, Aurélie Tchoghandjian, Stephane Honore, Dominique Figarella-Branger, Felix Bachmann, Heidi Lane, Diane Braguer. The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expression-dependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A183.