Symons, Marc

Brain and spinal tumors affect 1 in 1000 people by 25 years of age, and have diverse histological, biological, anatomical and dissemination characteristics. A mortality of 30–40% means the majority are cured, although two-thirds have life-long disability, linked to accumulated brain injury that is acquired prior to diagnosis, and after surgery or chemo-radiotherapy. Only four drugs have been licensed globally for brain tumors in 40 years and only one for children. Most new cancer drugs in clinical trials do not cross the blood–brain barrier (BBB). Techniques to enhance brain tumor drug delivery are explored in this review, and cover those that augment penetration of the BBB, and those that bypass the BBB. Developing appropriate delivery techniques could improve patient outcomes by ensuring efficacious drug exposure to tumors (including those that are drug-resistant), reducing systemic toxicities and targeting leptomeningeal metastases. Together, this drug delivery strategy seeks to enhance the efficacy of new drugs and enable re-evaluation of existing drugs that might have previously failed because of inadequate delivery. A literature review of repurposed drugs is reported, and a range of preclinical brain tumor models available for translational development are explored.

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 BACKGROUND Glioblastomas show marked intra- and inter-tumor heterogeneity and are strongly resistant to both radio- and chemo-therapy, which are standard therapeutic modalities for this tumor. MicroRNAs (miRNAs) have the potential to serve as effective therapeutics for glioblastoma as they modulate the activity of multiple signaling pathways. METHODS Glioblastoma cultures were transfected with miR-34a or control miRNA mimics to assess biological function and therapeutic potential in vitro. miR-34a was packaged into bacterially-derived nanocells and administered intravenously for delivery to orthotopic patient-derived glioblastoma xenografts in mice. RESULTS Overexpression of miR-34a strongly reduced the activation status of the three core signaling networks that have been found to be deregulated in the vast majority of glioblastoma tumors, the receptor tyrosine kinase, p53 and Rb networks. miR-34a transfection also inhibited the survival of multiple established glioblastoma cell lines as well as primary patient-derived xenograft cultures representing the proneural, mesenchymal and classical subtypes. Transfection of miR-34a synergized with temozolomide (TMZ) in in vitro cultures of glioblastoma cells with primary TMZ sensitivity, primary TMZ resistance and acquired TMZ resistance. Intravenous administration of bacterially-derived nanocells carrying miR-34a strongly enhanced TMZ sensitivity in an orthotopic patient-derived xenograft mouse model of glioblastoma. CONCLUSIONS miR-34a strongly sensitizes a wide range of glioblastoma cell cultures to TMZ, suggesting that combination therapy of TMZ with miR-34a may serve as a novel therapeutic approach for the treatment of glioblastoma tumors. Bacterially-derived nanocells are an effective vehicle for the delivery of miR-34a to glioblastoma tumors.

Abstract Therapeutic resistance stemming from inter and intra-tumoral heterogeneity is a significant impediment towards development of effective therapeutics for glioblastoma. We hypothesized that microRNAs can potentially counteract resistance emanating from such heterogeneity as they simultaneously modulate the expression of multiple proteins. We identified microRNA-34a as a unique microRNA which modulates multiple oncoproteins in GBM using two different in silico approaches. We investigated the therapeutic effects of microRNA-34a in three primary patient-derived xenografts (PDX) representing classical (GBM6), proneural (GBM118) and mesenchymal (GBM118) subtypes; four established cell lines (T98G, U251, A172, LN229) and two cell lines with acquired resistance to temozolomide (A172-TR, LN229-TR) in vitro. Glioblastoma cell cultures showed variable responses to temozolomide but microRNA-34a inhibited proliferation in all cell cultures. Furthermore, microRNA-34a also sensitized all tested cell lines to temozolomide (combination index < 0.8, p=.03) and radiation treatment (dose enhancement factor 1.7–2.2, p=0.02). Mechanistically, microRNA 34a down-regulates at least six distinct therapeutic resistance proteins. Importantly, these resistance proteins are expressed in distinct spatial niches and are prognostic for patient survival based on our analysis of the cancer genome atlas (TCGA) data. For in vivo delivery of microRNA-34a, we utilized nanocells which are derived from genetically modified bacteria, loaded with microRNA-34a and tagged with a bispecific antibody targeting EGFR. Nanocells were injected intravenously while temozolomide was administered by oral gavage in an orthotopic PDX model. We confirmed delivery of microRNA-34a to tumor by observing down-regulation of cMet and phosphorylated Akt in treated mice. Importantly, microRNA-34a nanocells resulted in significant reduction in tumor growth (p=0.021), increased survival (p<0.001) with microRNA-34a monotherapy and synergy in combination with temozolomide in vivo. Taken together, our results suggest that delivery of miR-34a may be a powerful new adjuvant for the treatment of glioblastoma in combination with temozolomide that can mitigate both inter- and intra-tumor heterogeneity.

Abstract Glioblastoma Multiforme (GBM) is the most common tumor of the CNS with a median survival of approximately 15 months. The highly aggressive invasion of malignant cells into the surrounding normal brain tissue renders complete surgical resection impossible, increases therapeutic resistance, and virtually assures tumor recurrence. We have previously demonstrated that TROY (TNFRSF19) has an important role in GBM invasion and resistance. TROY expression is correlated with glial tumor grade and inversely correlated with patient survival. Increased TROY expression stimulated cell migration/invasion in vitro and in vivo and confers resistance to both ionizing radiation- and temozolomide-induced apoptosis via activation of Akt and NF-κB. These data indicate that TROY stimulated migration/invasion is associated with signaling pathways that also increase survival and therapeutic resistance, however, the mechanistic basis of this signaling remains unclear. To investigate the mechanism by which TROY induces GBM cell invasion, we performed immunoprecipitation of the TROY receptor coupled with MALDI-TOF and MS/MS analysis and identified PDZ-RhoGEF as a binding partner for TROY. We validated the interaction of TROY with PDZ-RhoGEF by immunoprecipitation and western blotting, and demonstrated that PDZ-RhoGEF can exchange for both RhoA and RhoC in glioma cells and is implicated in GBM cell invasion, proliferation, and survival. In order to assess the specific role of PDZ-RhoGEF in TROY signaling, we examined the effects of PDZ-RhoGEF on TROY-mediated activation of NF-κB. While overexpression of TROY alone strongly stimulated NF-κB transcriptional activity in serum-free conditions, increased expression of PDZ-RhoGEF alone had no effect on NF-κB activity. Co-transfection of PDZ-RhoGEF with TROY synergistically promoted NF-κB activation. Mutation of the TRAF binding domain (TBD) in the cytoplasmic domain of TROY abolished its capacity to activate NF-κB, even in the presence of PDZ-RhoGEF, suggesting that the TBD is important for TROY induced NF-κB activation. Furthermore, knockdown of PDZ-RhoGEF by shRNA attenuated NF-κB activation in TROY expressing cells, blocked TROY mediated invasion of primary GBM xenograft cells and increased sensitivity to temozolomide. Collectively, these data indicate that PDZ-RhoGEF plays an important role in TROY signaling and provides insights into a potential node of vulnerability to limit GBM cell invasion and decrease therapeutic resistance. Citation Format: Zonghui Ding, Alison Roos, Jean Kloss, Harshil Dhruv, Marc Symons, Nhan L. Tran, Joseph C. Loftus. PDZ-RhoGEF functions as a critical signaling effector for TROY [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1878. doi:10.1158/1538-7445.AM2017-1878

Abstract Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. Despite surgical removal followed by concomitant radiation and chemotherapy with the alkylating agent temozolomide, GB tumors develop treatment resistance and ultimately recur. Impaired response to treatment occurs rapidly, conferring a median survival of just fifteen months. Thus, it is necessary to identify the genetic and signaling mechanisms that promote tumor resistance to develop targeted therapies to combat this refractory disease. Previous observations indicated that SGEF (ARHGEF26), a RhoG-specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and plays a role in promoting TWEAK-Fn14–mediated glioma invasion. Here, further investigation revealed an important role for SGEF in glioma cell survival. SGEF expression is upregulated by TWEAK-Fn14 signaling via NF-κB activity while shRNA-mediated reduction of SGEF expression sensitizes glioma cells to temozolomide-induced apoptosis and suppresses colony formation following temozolomide treatment. Nuclear SGEF is activated following temozolomide exposure and complexes with the DNA damage repair (DDR) protein BRCA1. Moreover, BRCA1 phosphorylation in response to temozolomide treatment is hindered by SGEF knockdown. The role of SGEF in promoting chemotherapeutic resistance highlights a heretofore unappreciated driver, and suggests its candidacy for development of novel targeted therapeutics for temozolomide-refractory, invasive GB cells. Implication: SGEF, as a dual process modulator of cell survival and invasion, represents a novel target for treatment refractory glioblastoma. Mol Cancer Res; 14(3); 302–12. ©2016 AACR.

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