MacCarthy, Brian

Glioblastoma (GBM), a highly invasive and vascular malignancy is shown to rapidly develop resistance and evolve to a more invasive phenotype following bevacizumab (Bev) therapy. Rho Guanine Nucleotide Exchange Factor proteins (RhoGEFs) are mediators of key components in Bev resistance pathways, GBM and Bev-induced invasion. To identify GEFs with enhanced mRNA expression in the leading edge of GBM tumours, a cohort of GEFs was assessed using a clinical dataset. The GEF βPix/COOL-1 was identified, and the functional effect of gene depletion assessed using 3D-boyden chamber, proliferation, and colony formation assays in GBM cells. Anti-angiogenic effects were assessed in endothelial cells using tube formation and wound healing assays. In vivo effects of βPix/COOL-1-siRNA delivered via RGD-Nanoparticle in combination with Bev was studied in an invasive model of GBM. We found that siRNA-mediated knockdown of βPix/COOL-1 in vitro decreased cell invasion, proliferation and increased apoptosis in GBM cell lines. Moreover βPix/COOL-1 mediated endothelial cell migration in vitro. Mice treated with βPix/COOL-1 siRNA-loaded RGD-Nanoparticle and Bev demonstrated a trend towards improved median survival compared with Bev monotherapy. Our hypothesis generating study suggests that the RhoGEF βPix/COOL-1 may represent a target of vulnerability in GBM, in particular to improve Bev efficacy.

Abstract Glioblastoma (GBM), a highly invasive primary brain tumour, remains an incurable disease (1). Angiogenesis, the formation of new blood vessels, is a defining feature of GBM (2). Targeting GBM angiogenesis with Bevacizumab (Bev) is associated with improved progression free survival, but may also significantly enhance local tumour invasion (3). RhoGTPases and their activators, guanine nucleotide exchange factors (GEFs), play central roles in the invasive process (4). Herein, we sought to identify and target GEFs of importance in meditating GBM invasion with a view to improving Bev response. We report a novel strategy by which GBM tumours invade and proliferate via overexpression of the GEF beta-PIX which was shown to be increased at the invasive edge in 74% of GBM tumours assessed (n = 19) compared with tumour core (5). We have further shown that siRNA-mediated knockdown of beta-PIX in GBM patient-derived xenograft cell cultures and cell lines resulted in decreased cell invasion in 3D, cell proliferation and survival assays in vitro. Furthermore, we have uncovered a role for beta-PIX expression in endothelial cell function, as knockdown of beta-PIX inhibits HUVEC cell migration in vitro. To interrogate in vivo effects, we established an imagable orthotopic model of invasive GBM in mice, by stereotactically implanting GBM cells stably overexpressing firefly luciferase (U87R-GFP-luc2). beta-PIX-(human and mouse) specific siRNA or scrambled control siRNA (2 × 10 μg i.c.) were delivered to tumours using an alphaVbeta3 integrin targeting-(RGD peptide conjugated)-nanoparticle (InVivoPlex Aparna Bio Corp, Rockville, MD). Animals were treated with- or without- Bev (6 × 10mg/kg i.p. over 12 days). Initial data has shown that animals treated with beta-PIX siRNA nanoparticles in combination with Bev had an improved median survival of 100 days compared with Bev- alone treated mice (median: 88 days). Our data may support the future clinical use of a beta-PIX-targeting nanoparticle therapeutic, to improve Bev outcomes in GBM. 1. Kohler BA et al J Natl Cancer Inst. 2011; 103, 714-736 2. Jain RK et al Nat Rev Neurosci. 2007; 8(8):610-22 3. Norden AD et al Neurology 2008; 70: 779-787 4. Murray D et al Brit J Can 2014; 110(5):1307-15 5. Hoelzinger DB et al Neoplasia 2005; 7(1): 7-16 Citation Format: David W. Murray, Philip O'Halloran, Monika Jarzabek, Brian MacCarthy, Jann N. Sarkaria, Raymond M. Schiffelers, Marc Symons, Annette T. Byrne. Pre-clinical assessment of a novel anti-invasion nanoparticle therapeutic in combination with bevacizumab for the treatment of glioblastoma. [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 5093. doi:10.1158/1538-7445.AM2015-5093