Orthotopic Glioblastoma Xenografts Research Articles

Novel Semi-Replicative Retroviral Vector Mediated Double Suicide Gene Transfer Enhances Antitumor Effects in Patient-Derived Glioblastoma Models.

As glioblastomas are mostly localized infiltrative lesions, gene therapy based on the retroviral replicating vector (RRV) system is considered an attractive strategy. Combinations of multiple suicide genes can circumvent the limitations associated with each gene, achieving direct and synergistic cytotoxic effects, along with bystander cell killing. In this study, we constructed a semi-and pseudotyped-RRV (sp-RRV) system harboring two suicide genes—herpes simplex virus type 1 thymidine kinase (TK) and yeast cytosine deaminase (CD)—to verify the dissemination and antitumor efficacy of our sp-RRV system (spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD) in seven patient-derived glioblastoma stem-like cells (GSCs). Flow cytometry and high-content analysis revealed a wide range of transduction efficiency and good correlation between the delivery of therapeutic genes and susceptibility to the prodrugs ganciclovir and 5-fluorocytosine in patient-derived GSCs in vitro. Intra-tumoral delivery of spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD, combined with prodrug treatment, synergistically inhibited cell proliferation and angiogenesis while increasing apoptosis and the depletion of tumor-associated macrophages in orthotopic glioblastoma xenografts. Genomic profiling of patient-derived GSCs revealed that the key genes preventing sp-RRV infection and transmission were associated with cell adhesion, migration, development, differentiation, and proliferation. This is the first report demonstrating that a novel sp-RRV-mediated TK/CD double suicide gene transfer system has high oncolytic power against extremely heterogeneous and treatment-refractory glioblastomas.

Speckle modulation enables high-resolution wide-field human brain tumor margin detection and in vivo murine neuroimaging

Current in vivo neuroimaging techniques provide limited field of view or spatial resolution and often require exogenous contrast. These limitations prohibit detailed structural imaging across wide fields of view and hinder intraoperative tumor margin detection. Here we present a novel neuroimaging technique, speckle-modulating optical coherence tomography (SM-OCT), which allows us to image the brains of live mice and ex vivo human samples with unprecedented resolution and wide field of view using only endogenous contrast. The increased visibility provided by speckle elimination reveals white matter fascicles and cortical layer architecture in brains of live mice. To our knowledge, the data reported herein represents the highest resolution imaging of murine white matter structure achieved in vivo across a wide field of view of several millimeters. When applied to an orthotopic murine glioblastoma xenograft model, SM-OCT readily identifies brain tumor margins with resolution of approximately 10 μm. SM-OCT of ex vivo human temporal lobe tissue reveals fine structures including cortical layers and myelinated axons. Finally, when applied to an ex vivo sample of a low-grade glioma resection margin, SM-OCT is able to resolve the brain tumor margin. Based on these findings, SM-OCT represents a novel approach for intraoperative tumor margin detection and in vivo neuroimaging.

Abstract 2321: Dual-function of CD27-CD70 costimulatory signal in CAR T cell therapy

Abstract Purpose: To investigate the function of co-stimulatory molecule CD27 on the antitumor efficacy of chimeric antigen receptor (CAR) T cells. Experimental Design: IL13 receptor α2 (IL13Rα2)-targeted CAR T cell products from glioblastoma (GBM) patients were characterized for CD27 surface expression and proliferation potential against recursive tumor challenge. Isolated CD27+ and CD27- fractions of CAR T cells were then evaluated for their antitumor potency against orthotopic GBM xenografts, as well as their molecular characteristics before and after tumor stimulation. Further, T cells were engineered to express a CAR construct together with a constitutively-expressed CD27 to investigate the role of continuous CD27 costimulation on CAR T cell maintenance and effector potency. Results: CD27 expression on CAR T cell products was correlated with their proliferation capacity against recursive tumor stimulation. Isolated CD27+ CAR T cells outperformed CD27- and unsorted cells in mediating long-term tumor eradication. CD27+ CAR T cells exhibited memory-associated genetic signatures, and were less exhausted following in vitro and in vivo tumor stimulation, both phenotypically and functionally. Moreover, CD27 also sensitized CAR T cells to target low antigen-expressing tumors. When targeting tumor cells expressing CD70 (CD27 ligand), CD27 on CAR T cells became rapidly down-regulated, suggesting a transient CD27-CD70 interaction. The advantageous effector function of CD27+ CAR T cells was diminished by blocking the CD27-CD70 interaction with CD70-targeting antibodies. CD70 is also expressed on CAR T cells, displaying a mutually exclusive expression pattern with CD27. Extended in vitro culture resulted in a phenotypic switch of CAR T cells from CD27+CD70- to CD27-CD70+. Surprisingly, constitutive expression of CD27 (CD27-cons) greatly impaired the effector activity of CAR T cells. These cells were able to eliminate tumor cells in vitro, but failed to mediate potent antitumor effect in vivo. Further characterization showed an early exhaustion phenotype for CD27-cons CAR T cells during in vitro expansion. Additionally, CD27-cons CAR T cells were also prone to activation-induced T cell apoptosis especially against high antigen-expressing targets. Conclusion: These studies demonstrate a dual-function of CD27 costimulation for CAR T cell antitumor activity. In the enriched CD27+ CAR T cell subset, the transient CD27-CD70 interaction with tumor cells resulted in enhanced antitumor potency. By contrast, constitutively expressed CD27 drove CAR T cell exhaustion and induced apoptosis. These results illustrate the critical role of CD27 in mediating potent CAR T cell therapeutic efficacy, and suggest the potential of CD27 as a marker for the functional quality of CAR T cell products. Citation Format: Dongrui Wang, Alfonso Brito, Darya Alizadeh, Renate Starr, Brenda Aguilar, Behnam Badie, Stephen J. Forman, Christine E. Brown. Dual-function of CD27-CD70 costimulatory signal in CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2321.

Abstract 3481: Roles of tumor suppressor candidate 2 (TUSC2) in glioblastoma progression and gliomagenesis

Abstract Tumor suppressor candidate 2 (TUSC2, also known as FUS1) was identified as a candidate tumor suppressor gene located in a region on chromosome 3p21.3 that undergoes allelic loss in lung and breast cancers. Loss of TUSC2 expression has been reported in various cancers and is associated with poor survival. Evidence to date indicates that TUSC2 behaves as a tumor suppressor in lung cancer; however, its role as a tumor suppressor in other tumor types has not been fully established. Since the mechanism for gliomagenesis is still unclear, we investigated the role of TUSC2 in the development and progression of glioblastoma (GBM), the most common and deadliest brain cancer in adults. Here, we found that forced TUSC2 expression suppressed neurosphere-forming capability of glioma stem cells, regardless of molecular subtypes. Forced expression of TUSC2 in GBM cell lines inhibited their ability to form colonies and neurospheres. To further determine whether TUSC2 plays a tumor suppressive role in GBM, we knocked down TUSC2 expression in TUSC2-expressing GBM cells using siRNA and CRISPR/Cas9, and found TUSC2 knockdown to significantly enhance neurosphere formation of GBM cells. Using an orthotopic GBM xenograft mouse model, we further observed that CRISPR/Cas9-mediated TUSC2 knockout significantly promoted the intracranial growth of GBM tumors. To gain inisghts into the mechanisms underlying TUSC2’s tumor suppressive function in GBM, we conducted RNA-Seq using control and TUSC2-knockout GBM cell lines, and identified a number of genes whose expression was altered in response to TUSC2 loss. Ongoing studies are being conducted to validate idenified genes, and elucidate their involvement in gliomagenesis and GBM progression. Furthermore, we speculated that TUSC2 may interact with cellular proteins leading to tumor suppression. To test this hypothesis, we conducted protein interactome analysis using immunoprecipitaton followed by mass spectrometry in which cell lysates from GBM cells and human astrocytes (a cell-of-origin for GBM) were used. This study has identified approximately 40 proteins that differentially interact with TUSC2 in GBM cells versus human astrocytes. Roles of these TUSC2-interacting proteins in GBM suppression and gliomagenesis are being examined in onging studies. Finally, we have generated conditional TUSC2-knockout mice to further address the role that TUSC2 plays in gliomagenesis. Collectively, these findings support a novel role that TUSC2 plays in GBM progression and gliomagenesis, thereby advancing our understanding of GBM pathobiology. Citation Format: Tadas K. Rimkus, Dongqin Zhu, Richard L. Carpenter, Ivy Paw, Austin Arrigo, Sherona Sirkisoon, Daniel Doheny, Noah Aguayo, Jingyun Lee, Guangxu Jin, Eric Spooner, Boris Pasche, Waldemar Debinski, Hui-Wen Lo. Roles of tumor suppressor candidate 2 (TUSC2) in glioblastoma progression and gliomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3481.

Long-term treatment with valganciclovir improves lentiviral suicide gene therapy of glioblastoma.

Suicide gene therapy for malignant gliomas has shown encouraging results in the latest clinical trials. However, prodrug application was most often restricted to short-term treatment (14 days), especially when replication-defective vectors were used. We previously showed that a substantial fraction of herpes simplex virus thymidine kinase (HSV-TK) transduced tumor cells survive ganciclovir (GCV) treatment in an orthotopic glioblastoma (GBM) xenograft model. Here we analyzed whether these TK+ tumor cells are still sensitive to prodrug treatment and whether prolonged prodrug treatment can enhance treatment efficacy. Glioma cells positive for TK and green fluorescent protein (GFP) were sorted from xenograft tumors recurring after suicide gene therapy, and their sensitivity to GCV was tested in vitro. GBM xenografts were treated with HSV-TK/GCV, HSV-TK/valganciclovir (valGCV), or HSV-TK/valGCV + erlotinib. Tumor growth was analyzed by MRI, and survival as well as morphological and molecular changes were assessed. TK-GFP+ tumor cells from recurrent xenograft tumors retained sensitivity to GCV in vitro. Importantly, a prolonged period (3 mo) of prodrug administration with valganciclovir (valGCV) resulted in a significant survival advantage compared with short-term (3 wk) application of GCV. Recurrent tumors from the treatment groups were more invasive and less angiogenic compared with primary tumors and showed significant upregulation of epidermal growth factor receptor (EGFR) expression. However, double treatment with the EGFR inhibitor erlotinib did not increase therapeutic efficacy. Long-term treatment with valGCV should be considered as a replacement for short-term treatment with GCV in clinical trials of HSV-TK mediated suicide gene therapy.

Quantitative histopathologic assessment of perfusion MRI as a marker of glioblastoma cell infiltration in and beyond the peritumoral edema region.

Conventional MRI fails to detect regions of glioblastoma cell infiltration beyond the contrast-enhanced T1 solid tumor region, with infiltrating tumor cells often migrating along host blood vessels. To quantitatively and qualitatively analyze the correlation between perfusion MRI signal and tumor cell density in order to assess whether local perfusion perturbation could provide a useful biomarker of glioblastoma cell infiltration. Animal model. Mice bearing orthotopic glioblastoma xenografts generated from a patient-derived glioblastoma cell line. 7T perfusion images acquired using a high signal-to-noise ratio (SNR) multiple boli arterial spin labeling sequence were compared with conventional MRI (T1 /T2 weighted, contrast-enhanced T1 , diffusion-weighted, and apparent diffusion coefficient). Immunohistochemistry sections were stained for human leukocyte antigen (probing human-derived tumor cells). To achieve quantitative MRI-tissue comparison, multiple histological slices cut in the MRI plane were stacked to produce tumor cell density maps acting as a "ground truth." Sensitivity, specificity, accuracy, and Dice similarity indices were calculated and a two-tailed, paired t-test used for statistical analysis. High comparison test results (Dice 0.62-0.72, Accuracy 0.86-0.88, Sensitivity 0.51-0.7, and Specificity 0.92-0.97) indicate a good segmentation for all imaging modalities and highlight the quality of the MRI tissue assessment protocol. Perfusion imaging exhibits higher sensitivity (0.7) than conventional MRI (0.51-0.61). MRI/histology voxel-to-voxel comparison revealed a negative correlation between tumor cell infiltration and perfusion at the tumor margins (P = 0.0004). These results demonstrate the ability of perfusion imaging to probe regions of low tumor cell infiltration while confirming the sensitivity limitations of conventional imaging modalities. The quantitative relationship between tumor cell density and perfusion identified in and beyond the edematous T2 hyperintensity region surrounding macroscopic tumor could be used to detect marginal tumor cell infiltration with greater accuracy. 1 Technical stage: 2 J. Magn. Reson. Imaging 2019;50:529-540.

OKN-007 Increases temozolomide (TMZ) Sensitivity and Suppresses TMZ-Resistant Glioblastoma (GBM) Tumor Growth

Treatment of glioblastoma (GBM) remains a challenge using conventional chemotherapy, such as temozolomide (TMZ), and is often ineffective as a result of drug resistance. We have assessed a novel nitrone-based agent, OKN-007, and found it to be effective in decreasing tumor volumes and increasing survival in orthotopic GBM xenografts by decreasing cell proliferation and angiogenesis and increasing apoptosis. In this study, we assessed combining OKN-007 with TMZ in vivo in a human G55 GBM orthotopic xenograft model and in vitro in TMZ-resistant and TMZ-sensitive human GBM cell lines. For the in vivo studies, magnetic resonance imaging was used to assess tumor growth and vascular alterations. Percent animal survival was also determined. For the in vitro studies, cell growth, IC50 values, RNA-seq, RT-PCR, and ELISA were used to assess growth inhibition, possible mechanism-of actions (MOAs) associated with combined OKN-007 + TMZ versus TMZ alone, and gene and protein expression levels, respectively. Microarray analysis of OKN-007–treated rat F98 glioma tumors was also carried out to determine possible MOAs of OKN-007 in glioma-bearing animals either treated or not treated with OKN-007. OKN-007 seems to elicit its effect on GBM tumors via inhibition of tumorigenic TGF-β1, which affects the extracellular matrix. When combined with TMZ, OKN-007 significantly increases percent survival, decreases tumor volumes, and normalizes tumor blood vasculature in vivo compared to untreated tumors and seems to affect TMZ-resistant GBM cells possibly via IDO-1, SUMO2, and PFN1 in vitro. Combined OKN-007 + TMZ may be a potentially potent treatment strategy for GBM patients.

Evaluation of four different small animal radiation plans on tumour and normal tissue dosimetry in a glioblastoma mouse model.

Small animal radiotherapy research platforms such as XStrahl's SARRP enable more precise irradiation of tumours and normal tissues in pre-clinical models of cancer. Using an orthotopic G7 glioblastoma xenograft model we studied the impact of four different radiotherapy plans on tumour and normal tissue dosimetry. Plans were created using four different approaches (single beam, parallel opposed pair, single plane arcs, couch rotation arcs) and dose volume histograms (DVH) for the tumour and the relevant organs at risk (OARs) (mouth, ipsilateral brain, contralateral brain, brain stem) were compared for a sample mouse subject. To evaluate the accuracy of delivery, treatment plans were recreated in solid-water phantoms and delivered to radiochromic film. Favourable tumour dosimetry was achieved by all plans. DVH analysis showed that different plans could be used to spare specific OARs depending on the objectives of the study. The delivery accuracy of the various treatments was better than 2%/2mm (dose difference/distance to agreement) in terms of global γ analysis. Small animal radiotherapy research platforms are an exciting addition to the pre-clinical research environment. Such systems improve the conformality of irradiation of tumours and OARs while maintaining a high degree of accuracy and enable investigators to optimise experiments in terms of tumour coverage and inclusion or exclusion of relevant OARs. This study confirms the utility of the SARRP in terms of the accuracy of plan delivery, and informs decisions on treatment planning to optimise the clinical relevance and scientific value of experiments.

Ars2 promotes cell proliferation and tumorigenicity in glioblastoma through regulating miR-6798-3p

Arsenic resistance protein 2 (Ars2) is a component of the nuclear RNA cap-binding complex (CBC) that is important for some microRNA biogenesis and it is critical for cell proliferation and tumorigenicity. However, mechanism of Ars2-regulated cellular proliferation and tumorigenicity in glioblastoma has not been fully understood. Western blotting was used to detect the expressions of Ars2, p53, p21, and cleavage/activation of caspases-3 (C-Caspase 3). Microarray and Quantitative Real-time PCR (qRT-PCR) were performed to identify the Ars2-regulated microRNAs. Apoptosis assessed by flow cytometry analysis was used to evaluate the role of Ars2 in cells proliferation. The lentivirus-mediated gene knockdown approach was conducted to determine the function of Ars2. The orthotopic glioblastoma xenograft was used to demonstrate the role of Ars2 in glioblastoma growth in vivo. The high expression of Ars2 was observed in several glioblastoma cell lines and was significantly associated with poorer overall survival. Importantly, the overexpression of Ars2 promoted cell proliferation and colony formation in glioblastoma cells, whereas the depletion of Ars2 inhibited cell proliferation, colony formation, and tumor growth. Mechanistic study revealed that knockdown of Ars2 reduced the expression levels of miR-6798-3p, which was responsible for the up-regulation of p53 and p21, leading to apoptosis. Furthermore, the knockdown of Ars2 suppressed tumor growth in orthotopic glioblastoma xenograft model and significantly prolonged the survival time of the tumor-bearing mice. These findings identify a critical role for Ars2 in regulation of proliferation and tumorigenicity in glioblastoma and suggest that Ars2 could be a critical therapeutic target for glioblastoma intervention.

Effective and Targeted Human Orthotopic Glioblastoma Xenograft Therapy via a Multifunctional Biomimetic Nanomedicine.

Glioblastoma multiforme (GBM) is a fatal central nervous system tumor without effective treatment. Chemotherapeutic agents are mainstays in the treatment of glioblastoma. However, the effectiveness of these is seriously hindered by poor blood-brain-barrier (BBB) penetrance and tumor targeting, together with short biological half-life. Improved chemotherapy is thus urgently needed for GBM. Multifunctional nanoparticle delivery systems offer much promise in overcoming current limitations. Accordingly, a multifunctional biomimetic nanomedicine is developed by functionalizing the surface of red blood cell membranes (RBCms) with angiopep-2 and loading pH-sensitive nanoparticles (polymer, doxorubicin (Dox), and lexiscan (Lex)) using the functionalized cell membrane to generate the novel nanomedicine, Ang-RBCm@NM-(Dox/Lex). The studies toward orthotopic U87MG human glioblastoma tumor-bearing nude mice show that the Ang-RBCm@NM-(Dox/Lex) nanomedicine has much improved blood circulation time, superb BBB penetration, superior tumor accumulation and retention. Moreover, effective suppression of tumor growth and significantly improved medium survival time are also observed after Ang-RBCm@NM-(Dox/Lex) treatment. The results show that this biomimetic nanoplatform can serve as a flexible and powerful system for GBM treatment which can be readily adapted for the treatment of other central nervous system (CNS) disorders.

An Efficient Bivalent Cyclic RGD-PIK3CB siRNA Conjugate for Specific Targeted Therapy against Glioblastoma In Vitro and In Vivo

The PI3K-AKT-mTOR-signaling pathway is frequently activated in glioblastoma (GBM). Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB)/p110β (a PI3K catalytic isoform) by RNAi substantially suppresses GBM growth with less toxicity to normal astrocytes. However, insufficient and non-specific small interfering RNA (siRNA) delivery may limit the efficacy of RNAi-based therapies against GBM. Here we prepared a novel methoxy-modified PIK3CB siRNA molecule (siPIK3CB) that was covalently conjugated to a [cyclo(Arg-Gly-Asp-D-Phe-Lys)-Ahx]2-Glu-PEG-MAL (biRGD) peptide, which selectively binds to integrin αvβ3 receptors. The αvβ3-positive U87MG cell line was selected as a representative for GBM. An orthotopic GBM xenograft model based on luciferase-expressing U87MG was established and validated in vivo to investigate bio-distribution and anti-tumor efficacy of biRGD-siPIK3CB. In vitro, biRGD-siPIK3CB specifically entered and silenced PIK3CB expression in GBM cells in an αvβ3 receptor-dependent manner, thus inhibiting cell cycle progression and migration and enhancing apoptosis. In vivo, intravenously injected biRGD-siPIK3CB substantially slowed GBM growth and prolonged survival by reducing tumor viability with silencing PIK3CB expression. Furthermore, biRGD-siPIK3CB led to mild tubulointerstitial injury in the treatment of GBM without obvious hepatotoxicity, whereas co-infusion of Gelofusine obviously alleviated this injury without compromising anti-tumor efficacy. These findings revealed a great translational potential of biRGD-siPIK3CB conjugate as a novel molecule for GBM therapy.

Dendritic cell-mediated delivery of doxorubicin-polyglycerol-nanodiamond composites elicits enhanced anti-cancer immune response in glioblastoma

Glioblastoma (GBM) is the deadliest and most common type of primary brain tumor in adults with a grim prognosis despite multimodal treatments. Dendritic cell (DC)-based immunotherapy has emerged as a promising therapeutic modality for GBM, whose efficacy is nonetheless fundamentally undermined by GBM-induced immunosuppression. Inducing emission of damage associated molecular patterns (DAMPs) is a highly effective strategy to subvert tumor-associated immunosuppression. The present work was carried out to explore the idea of subverting the GBM immunosuppressive microenvironment through DC-mediated delivery of doxorubicin-polyglycerol-nanodiamond composites (Nano-DOX), a potent DAMPs inducer demonstrated by our previous study, and thereby eliciting enhanced DC-driven anti-GBM immune response. In the in-vitro work on human cell models, Nano-DOX-loaded DC were shown to be functionally viable and release cargo drug to co-cultured GBM cells (GC). Nano-DOX-treated GC displayed not only profuse DAMPs emission but also antigen release. Enhanced activation and acquisition and presentation of GC-derived antigen were then demonstrated in DC in co-culture with GC and Nano-DOX. Consistently, co-culture with GC and Nano-DOX also activated mouse bone marrow-derived DC (mDC) which in turn stimulated mouse spleen-derived lymphocytes which ultimately suppressed co-cultured GC. Next, athymic mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mDC and, 48 h later, spleen-derived lymphocytes. The presence of Nano-DOX, DAMPs emission and enhanced infiltration and activation of mDC and lymphocytes were detected in the GBM xenografts. Taken together, our results demonstrate the efficacy of DC-mediated delivery of Nano-DOX to stimulate GC immunogenicity and elicit anti-cancer immune response in the GBM. By this work, we present a novel approach with great application potential to subverting the GBM immunosuppressive microenvironment and to anti-GBM immunotherapy. Investigation has also been conducted probing the mechanisms by which Nano-DOX stimulates GC immunogenicity, which is described in a follow-up paper.

Abstract 1764: Neurovascular-targeting antibodies discovered using yeast biopanning enhance drug delivery and improve survival for glioblastoma

Abstract INTRODUCTION: Median survival for newly diagnosed glioblastoma (GBM) patients remains less than two years despite aggressive clinical interventions. Antibody-based GBM-targeting could improve brain delivery and accumulation of therapies, but it has been challenging to identify universal tumor cell antigens due to intra-tumor and inter-patient GBM heterogeneity. We hypothesized that antigens of the neurovascular extracellular matrix (nvECM) within GBM-disrupted blood-brain barrier (BBB) may localize and enhance efficacy of therapeutic agents. METHODS: Yeast surface display biopanning and high throughput screening identified Variable Lymphocyte Receptors (VLR, i.e. jawless vertebrate lamprey antibodies) that preferentially accumulate at nvECM. Lead VLR candidates were tested for tumor targeting via combination with chemotherapeutic doxorubicin (DOX) liposomes and assessed for anti-GBM effects in vitro and against orthotopic mouse xenografts in vivo. RESULTS: VLR clones were enriched for binding to mouse brain endothelial cell (bEnd.3 line) ECM with two rounds of biopanning, then screened for preferential binding to brain-derived ECM. Lead VLR clone P1C10 was chosen for further testing for nvECM specificity vs. fibroblast (3T3 line) ECM in a high throughput screen, high affinity (Kd=48±6.1 nM), and immunolabeling of normal mouse and human brain, and GBM samples. Clone P1C10 demonstrated tumor-specific targeting in orthotopic U87 GBM xenografts, with 50% more DOX fluorescence detected at GBM after intravenous injection of P1C10-DOX liposomes compared to control, non-specific VLR-DOX. Finally, administration of P1C10-DOX liposomes (15 mg/kg, i.p., 1/week) significantly improved survival of mice bearing U87 xenografts, compared to non-targeted VLR-DOX (Kaplan-Meier, p<0.01). CONCLUSIONS: We report combining yeast surface display biopanning and screening to identify VLRs that preferentially accumulate at disrupted nvECM of GBM-disrupted BBB, and demonstrate GBM-targeting specificity in vitro and in vivo with significant survival benefit. These results support the nvECM immune-targeting strategy to improve anti-GBM therapeutic efficacy, potentially improving survival and quality-of-life in brain cancer patients. Citation Format: Paul A. Clark, Benjamin J. Umlauf, Eric V. Shusta, John S. Kuo. Neurovascular-targeting antibodies discovered using yeast biopanning enhance drug delivery and improve survival for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1764.

Abstract 5825: Structure-based drug design to overcome temozolomide resistance in glioblastoma (GBM) through a dual inhibition of MGMT and base excision repair

Abstract The incidence of pediatric and adult brain tumors has continued to rise and temozolomide (TMZ) has remained the sole choice for chemotherapy in these cancers. TMZ alkylates the DNA to generate N7-methylguanine (~70%) and O6-methylguanine (~6-9%) lesions, with the latter being considered the cytotoxic residue. However, the N7-methylations can also contribute to cell killing by producing abasic sites, strand-breaks, and mutagenicity. Therefore, inhibiting the two major mechanisms of resistance to TMZ in brain cancers, namely, the MGMT DNA repair, which removes the O6-methyl groups, and the APE1 endonuclease, which cleaves the apurinic/apyrimidinic sites, is a logical choice for enhancing the alkylator therapy; incidentally, both these proteins are highly expressed in brain tumors. Pseudosubstrates for MGMT such as O6-BG + TMZ therapies suffer from a high level of bone marrow suppression and there is no clinically approved inhibitor for APE1. Recently, we discovered that nitroaspirin (NCX-4016) is a powerful and clinically relevant inhibitor of human MGMT for increasing the efficacy of alkylating agents (Proc of AACR 54, 2013;1094). Further extensive structure-activity relationship studies revealed that the 3-hydroxybenzyl nitrate moiety of NCX-4016 is essential for its MGMT inhibition activity. To develop a dual inhibitor of APE1 and MGMT, we designed a hybrid compound called MGAP-9 by combining 3-hydroxybenzyl nitrate with 7-nitroindole-2-carboxylic acid, a known potent, nontoxic, and cell-permeable APE1 inhibitor through an ester bond. MGAP-9 exhibited significant inhibition of MGMT activity and a corresponding decrease in protein levels in both concentrations- and time-dependent manner. Similarly, we observed potent inhibition of APE1 nuclease activity both in vitro and in tumor cells using a biotin-labeled oligonucleotide/polyacrylamide gel-electrophoresis assay. Inhibition of both targets occurred at pharmacologically achievable concentrations (5-25 µM). MGAP-9 exhibited more than 100-fold sensitization of various GBM cell lines towards the methylating agents such as TMZ and dacarbazine, but surprisingly not to BCNU or CCNU. A substantial increase in γ-H2AX further confirmed the DNA damage induced by MGAP-9 through a dual inhibition. In addition, the hybrid drug displayed potent antiangiogenic activity against human umbilical vein cells (HUVECs). Finally, MGAP-9 showed significant regression of tumor growth in mouse orthotopic glioblastoma xenograft models and did not elicit systemic toxicity at the therapeutic doses used. The findings suggest the clinical relevance of MGAP-9 and its usefulness in reducing the TMZ doses to curtail the marrow suppression in brain tumor therapy. Supported by CPRIT grants RP130266 and RP170207 to KSS. Citation Format: Hanumantha Rao Madala, Viswanath Arutla, Surendra Punganuru, Kalkunte Srivenugopal. Structure-based drug design to overcome temozolomide resistance in glioblastoma (GBM) through a dual inhibition of MGMT and base excision repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5825.

Bioreducible Polymeric Nanoparticles Containing Multiplexed Cancer Stem Cell Regulating miRNAs Inhibit Glioblastoma Growth and Prolong Survival.

Despite our growing molecular-level understanding of glioblastoma (GBM), treatment modalities remain limited. Recent developments in the mechanisms of cell fate regulation and nanomedicine provide new avenues by which to treat and manage brain tumors via the delivery of molecular therapeutics. Here, we have developed bioreducible poly(β-amino ester) nanoparticles that demonstrate high intracellular delivery efficacy, low cytotoxicity, escape from endosomes, and promotion of cytosol-targeted environmentally triggered cargo release for miRNA delivery to tumor-propagating human cancer stem cells. In this report, we combined this nanobiotechnology with newly discovered cancer stem cell inhibiting miRNAs to develop self-assembled miRNA-containing polymeric nanoparticles (nano-miRs) to treat gliomas. We show that these nano-miRs effectively intracellularly deliver single and combination miRNA mimics that inhibit the stem cell phenotype of human GBM cells in vitro. Following direct intratumoral infusion, these nano-miRs were found to distribute through the tumors, inhibit the growth of established orthotopic human GBM xenografts, and cooperatively enhance the response to standard-of-care γ radiation. Co-delivery of two miRNAs, miR-148a and miR-296-5p, within the bioreducible nano-miR particles enabled long-term survival from GBM in mice.

Discovery and Optimization of 2-Amino-4-methylquinazoline Derivatives as Highly Potent Phosphatidylinositol 3-Kinase Inhibitors for Cancer Treatment.

Increased phosphatidylinositol 3-kinase (PI3K) signaling is among the most common alterations in cancer, spurring intensive efforts to develop new cancer therapeutics that target this pathway. In this work, we discovered a series of novel 2-amino-4-methylquinazoline derivatives through a hybridization and subsequent scaffold hopping approach that were highly potent class I PI3K inhibitors. Lead optimization resulted in several promising compounds (e.g., 19, 20, 37, and 43) with nanomolar PI3K potencies, prominent antiproliferative activities, favorable PK profiles, and robust in vivo antitumor efficacies. More interestingly, compared with 19 and 20, 37 and 43 demonstrated improved brain penetration and in vivo efficacy in an orthotopic glioblastoma xenograft model. Furthermore, preliminary safety assessments including hERG channel inhibition, AMES, CYP450 inhibition, and single-dose toxicity were performed to characterize their toxicological properties.

Protein Toxin Chaperoned by LRP-1-Targeted Virus-Mimicking Vesicles Induces High-Efficiency Glioblastoma Therapy In Vivo.

Glioblastoma is a most intractable and high-mortality malignancy because of its extremely low drug accessibility resulting from the blood-brain barrier (BBB). Here, it is reported that angiopep-2-directed and redox-responsive virus-mimicking polymersomes (ANG-PS) (angiopep-2 is a peptide targeting to low-density lipoprotein receptor-related protein-1 (LRP-1)) can efficiently and selectively chaperone saporin (SAP), a highly potent natural protein toxin, to orthotopic human glioblastoma xenografts in nude mice. Unlike chemotherapeutics, free SAP has a low cytotoxicity. SAP-loaded ANG-PS displays, however, a striking antitumor activity (half-maximal inhibitory concentration, IC50 = 30.2 × 10-9 m) toward U-87 MG human glioblastoma cells in vitro as well as high BBB transcytosis and glioblastoma accumulation in vivo. The systemic administration of SAP-loaded ANG-PS to U-87 MG orthotopic human-glioblastoma-bearing mice brings about little side effects, effective tumor inhibition, and significantly improved survival rate. The protein toxins chaperoned by LRP-1-targeted virus-mimicking vesicles emerge as a novel and highly promising treatment modality for glioblastoma.

Truncated Glioma-Associated Oncogene Homolog 1 (tGLI1) Mediates Mesenchymal Glioblastoma via Transcriptional Activation of CD44.

The molecular pathways driving mesenchymal glioblastoma (GBM) are still not well understood. We report here that truncated glioma-associated oncogene homolog 1 (tGLI1) is a tumor-specific transcription factor that facilitates GBM growth, is enriched in the mesenchymal subtype of GBM and glioma stem cells (GSC), and promotes mesenchymal GSC by upregulating transcription of CD44. In an orthotopic GBM xenograft mouse model, tGLI1-overexpressing tumors grew more aggressively with increased proliferation and angiogenesis compared with control and GLI1-overexpressing xenografts. tGLI1 was highly expressed in GBM clinical specimens but undetectable in normal brains, whereas GLI1 was expressed in both tissues. A tGLI1 activation signature (tGAS) correlated with glioma grade, tumor angiogenesis, and poor overall survival, and GBMs with high tGAS were enriched with mesenchymal GBM/GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared with the monolayer culture; mesenchymal GSC expressed more tGLI1 than proneural GSC. Ectopic tGLI1 expression enhanced the ability of mesenchymal GSC to yield neurospheres in vitro and to form tumors in mouse brains. Selective tGLI1 knockdown reduced neurosphere formation of GBM cells. tGLI1 bound to and transactivated the promoter of the CD44 gene, a marker and mediator for mesenchymal GSC, leading to its expression. Collectively, these findings advance our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSC.Significance: These findings highlight the role of a tumor-specific gain-of-function transcription factor tGLI1 in mesenchymal glioma stem cell maintenance and mesenchymal GBM growth. Cancer Res; 78(10); 2589-600. ©2018 AACR.

Abstract B125: A novel BBB-permeable chemotherapeutic agent for the treatment of CNS tumors

Abstract Standard of care for glioblastoma multiforme (GBM) patients is surgical tumor resection, followed by radiation and chemotherapy with temozolomide. Unfortunately, 60% of newly diagnosed GBM patients express high levels of the DNA repair enzyme MGMT and are temozolomide-insensitive, and all patients eventually become refractory to treatment. The blood-brain barrier (BBB) remains an obstacle to adequate delivery of chemotherapeutic agents to brain tumors. Lipophilic drugs such as temozolomide and lomustine are able to cross the BBB based on passive diffusion; however, their tissue distribution also causes toxicity to critical systemic organs, such as the bone marrow, leading to dose-limiting toxicity. Therefore, BBB-permeable chemotherapeutic agents that are efficacious in temozolomide-insensitive and refractory patients are needed. The large amino acid transporter 1 (LAT1) is highly expressed on the BBB and in GBM and other malignant brain tumors such as diffuse intrinsic pontine glioma (DIPG), where it is associated with poor prognosis, but is undetectable in normal brain tissue. Targeting LAT1 transport, while avoiding interaction with other closely related transporters expressed on normal tissue such as LAT2, will target brain tumors and avoid uptake into healthy tissue. We report the generation of a novel molecule (QBS10072S) that combines a potent cytotoxic domain with the structural features of a selective LAT1 substrate. QBS10072S is 50-fold more selective for LAT1 vs. LAT2 in transport assays, and demonstrates significant in vitro cytotoxicity to LAT1-expressing GBM cell lines and minimal cytotoxicity to normal human astrocytes (LAT1-negative). Unlike temozolomide, QBS10072S is cytotoxic to cells with both high and low levels of MGMT expression. In orthotopic glioblastoma xenografts, QBS10072S causes significant delay in tumor growth and increase in mouse survival compared to vehicle. QBS10072S is well tolerated, with no toxicity seen in normal brain or the BBB. In summary, QBS10072S is a novel BBB-permeable chemotherapeutic agent with the potential to treat temozolomide-insensitive and recurrent GBM. Citation Format: Theodore P. Nicolaides, Tomoko Ozawa, Tsun Wen Yao, Michael Prados, Wolf-Nicolas Fischer, Bernd Jandeleit, Kerry Koller. A novel BBB-permeable chemotherapeutic agent for the treatment of CNS tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B125.

Restricted Delivery of Talazoparib Across the Blood–Brain Barrier Limits the Sensitizing Effects of PARP Inhibition on Temozolomide Therapy in Glioblastoma

Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1-3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2-M arrest in vitroIn vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp-/- versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b-/- mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood-brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735-46. ©2017 AACR.