GBM Growth Research Articles

ACT001 reduces the expression of PD-L1 by inhibiting the phosphorylation of STAT3 in glioblastoma.

ACT001, which is derived from an ancient anti-inflammatory drug, has been shown to cross the blood-brain barrier in preclinical studies and has demonstrated anti-glioblastoma (GBM) activity in clinical trials. However, its pharmacological potential for anti-GBM immune response modulation remains unclear. The chemical structure of ACT001 indicates that it may bind to STAT3 and thus modulate antitumor immune response.Methods: Bioinformatics and immunohistochemistry (IHC) were used to assess STAT3 and PD-L1 expression in gliomas. Western blotting, RT-PCR and immunofluorescence were used to detect PD-L1 and p-STAT3 expression in glioma cells exposed to ACT001. Chromatin immunoprecipitation, an ACT001-Biotin probe, and a dual-luciferase reporter assay were used to detect direct modulation. The in vivo efficacy of ACT001 was evaluated in GL261 murine glioma model. Survival analyses were conducted using the log-rank (Mantel-Cox) test.Results: Bioinformatic analysis of 1,837 samples from 4 public glioma datasets showed that STAT3 mRNA expression was correlated with the degree of malignancy and therapeutic resistance and that STAT3 mRNA expression was related to immunosuppression, leukocyte infiltration, and PD-L1 expression. IHC staining of 53 tissue samples confirmed that relatively high phosphorylated STAT3 and PD-L1 protein expression was associated with a relatively advanced World Health Organization (WHO) glioma grade. Next, we confirmed that ACT001 treatment reduced PD-L1 expression and STAT3 phosphorylation. An ACT001-biotin probe was used to verify that ACT001 bound to STAT3. We also demonstrated that STAT3 bound to the PD-L1 promoter. The inhibition of PD-L1 expression and STAT3 phosphorylation by ACT001 could be rescued by STAT3 overexpression. Additionally, ACT001 inhibited GBM growth and decreased PD-L1 expression in vivo. The expression of the M2 markers CD206 and CD163 was decreased, while that of the antitumor immune markers iNOS and IFNγ was increased by ACT001 in vivo.Conclusion: Our results demonstrate that STAT3 plays a key role in immunosuppression of glioma and is inhibited by ACT001. ACT001 inhibits PD-L1 transcription and modulates anti-tumor immune response in glioma bearing mice. These findings will help us to understand the mechanism of ACT001 in GBM therapy.

Abstract C112: A bad apple spoils the bunch: How environment drives tumor aggressiveness and resistance

Abstract Glioblastoma (GBM) is the most common and aggressive type of malignant brain tumor in adults in the United States. Current treatment options at diagnosis are multimodal and include surgical resection, radiation, and chemotherapy. While the aim of combined therapy approach to GBM is long term remission, it does so in only about 20% of patients. Because the current standard GBM treatment is unlikely to result in prolonged remission, there is a great effort in Nuero-Oncology research field to better understand the oncobiology of GBM and overcome tumor resistance. GBM arises from multiple cell type with neural stem-cell proprieties (GSCs). GBM molecular profile reveled 2 predominant subtypes: proneural (PN) and mesenchymal (MES). Tumor subtype has significant prognostic value, with GBM tumors with mesenchymal signature displaying a more aggressive phenotype. Furthermore, PN tumors tend to shift toward a MES phenotype upon recurrence or after radiation therapy. Emerging evidence indicates that this phenotypic change occurs, at least in part, due to direct conversion of one cell type to another rather than selection of pre-existing therapy-resistant tumor cells. GBM grows in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. Neuropeptides are a group of signaling messengers that function as neurotransmitters, paracrine regulators, and hormones to regulate exocrine and endocrine secretion and inflammation. Previously, neuropeptides have also been recognized as potent cellular growth factors for many cell types, including cancer cells. Extracellular vesicles (EVs) are critical mediators of intercellular communication between tumor cells and stromal cells in local and distant microenvironments. EVs play an essential role in both primary tumor growth and metastatic evolution. EVs contain bioactive molecules, such as nucleic acids, protein and lipids that can redirect the function of a recipient cell. Based on the regulatory function of neuropeptides we hypothesized that neuropeptides could be involved in intercellular communication between PN and MES GSCs within the tumor milieu. The aim of this project is the characterization of the neuropeptide profile of PN and MES GSCs and to investigate whether EVs could play a role in GSCs plasticity within the tumor milieu. Initially, a gene expression analysis from PN and MES GSCs was performed based on the data from a custom RNA array for human neuropeptides. After validation, five targets were selected to be tested in the intercellular communication mediated by EVs. In order to address the role of EVs in neuropeptide intercellular communication, EVs from condition medium from MES GSCs were isolated. Then, PN GSCs were treated with MES derived EVs (EVs-MES). After treatment, RNA purification was performed for real time PCR analysis. This work is the first to analyze the neuropeptide profile of GSCs. Our data show that PN and MES have a differential neuropeptide expression profile. Most interestingly is the fact that the treatment of PN cells with EVs-MES drove a shift in the neuropeptide expression in PN cells. Taking this data together we believe that neuropeptides could be one of the milieu factors linked to phenotype plasticity in GBM and could provide a new target for upcoming treatment options for GBM. Citation Format: Renata Fleming, Markus W Schweiger, Bakhos A Tannous. A bad apple spoils the bunch: How environment drives tumor aggressiveness and resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C112. doi:10.1158/1535-7163.TARG-19-C112

RETRACTED ARTICLE: Dissecting the role of novel EZH2 inhibitors in primary glioblastoma cell cultures: effects on proliferation, epithelial-mesenchymal transition, migration, and on the pro-inflammatory phenotype

BackgroundGlioblastoma (GBM) is the most lethal and aggressive malignant primary brain tumor in adults. After surgical resection of the tumor, the patient typically should be subjected to chemotherapy (temozolomide, TMZ) and concomitant radiotherapy. Since the TMZ treatment does not lead to complete remission and often develops resistance, the identification of efficacious therapeutics is strongly to pursue. Among the epigenetic players, the H3K27 methyltransferase (MT) EZH2 (enhancer of zeste homologue 2) has been found overexpressed or mutated in several human cancers including gliomas, and its overexpression is associated with poor outcome in GBM. Two EZH2 inhibitors (EZH2i), UNC1999 and GSK343, suppressed GBM growth in vitro and in vivo indicating that EZH2i can be potential drugs against GBM.ResultsTwo new EZH2i, MC4040 and MC4041, were designed, prepared, and tested by us to determine their effects in primary GBM cell cultures. MC4040 and MC4041 displayed single-digit micromolar inhibition of EZH2, 10-fold less potency against EZH1, and no activity towards other MTs. In primary GBM cells as well as in U-87 GBM cells, the two compounds reduced H3K27me3 levels, and dose- and time-dependently impaired GBM cell viability without inducing apoptosis and arresting the cell cycle in the G0/G1 phase, with increased p21 and p27 levels. In combination with TMZ, MC4040 and MC4041 displayed stronger, but not additive, effects on cell viability. The potent clinical candidate as EZH2i tazemetostat, alone or in combination with TMZ, exhibited a similar potency of inhibition of GBM cell growth when compared to MC4040 and MC4041. At the molecular level, MC4040 and MC4041 reduced the VEGFR1/VEGF expression, reversed the epithelial-mesenchymal transition (EMT), and hampered cell migration and invasion attenuating the cancer malignant phenotype. Treatment of GBM cells with MC4040 and MC4041 also impaired the GBM pro-inflammatory phenotype, with a significant decrease of TGF-β, TNF-α, and IL-6, joined to an increase of the anti-inflammatory cytokine IL-10.ConclusionsThe two novel EZH2i MC4040 and MC4041 impaired primary GBM cell viability, showing even stronger effects in combination with TMZ. They also weakened the aggressive malignant phenotype by reducing angiogenesis, EMT, cell migration/invasion and inflammation, thus they may be considered potential candidates against GBM also for combination therapies.

Reduced expression of proteolipid protein 2 increases ER stress-induced apoptosis and autophagy in glioblastoma.

Proteolipid protein 2 (PLP2) is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be highly expressed in many cancer types, but its importance in glioma progression is poorly understood. Using publicly available datasets (Rembrandt, TCGA and CGGA), we found that the expression of PLP2 was significantly higher in high‐grade gliomas than in low‐grade gliomas. We confirmed these results at the protein level through IHC staining of high‐grade (n = 56) and low‐grade glioma biopsies (n = 16). Kaplan‐Meier analysis demonstrated that increased PLP2 expression was associated with poorer patient survival. In functional experiments, siRNA and shRNA PLP2 knockdown induced ER stress and increased apoptosis and autophagy in U87 and U251 glioma cell lines. Inhibition of autophagy with chloroquine augmented apoptotic cell death in U87‐ and U251‐siPLP2 cells. Finally, intracranial xenografts derived from U87‐ and U251‐shPLP2 cells revealed that loss of PLP2 reduced glioma growth in vivo. Our results therefore indicate that increased PLP2 expression promotes GBM growth and that PLP2 represents a potential future therapeutic target.

DDIS-37. SMALL MOLECULE AUTOPHAGY ACTIVATORS IN GROWTH INHIBITION OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is one of the most lethal human tumors, and despite considerable advances in multimodality treatments consisting of surgical removal, radiation and chemotherapy, overall survival is 14 to 16 months after diagnosis. Therefore, there is a need to target non-apoptotic death pathways and inhibit pro-survival signaling mechanisms that contribute to both tumorigenesis and treatment resistance. Depending on tumor type and environment, autophagy modulation plays an important role in tumor cell survival and inhibition pathways. Increasing evidence supports that chemical modulation of autophagy inhibition and activation holds a therapeutic potential and these studies have led to the initiation of multiple clinical trials combining chemotherapeutic agents and autophagy inhibitors and activators for various cancer types. However, recent studies have also demonstrated a therapeutic potential for enhancers of autophagy in GBM. This clearly demonstrates the need for further investigation into the autophagy induced mechanism of GBM, and the development of better treatments. We have identified a new chemical entity VMY-BC-1 that showed promising effects in both serum and patient-derived glioma cell lines. Drug target engagement studies identified an autophagy induction protein, UNC-51 like kinase 1 (ULK1), as the main target of VMY-BC-1, in which VMY-BC-1 covalently bound to the protein’s active pocket and increased ULK1 phosphorylation.ULK1 is a component of the ULK1 complex (ULK1-ATG13-FIP200) that is essential for inducing autophagy, a lysosome-dependent mechanism of intracellular degradation for maintaining cellular homeostasis. Additionally, our molecule cross talks to the apoptotic and DNA damage pathways, which could potentially overcome treatment resistance.VMY-BC-1 is orally bioavailable, brain penetrant, and inhibits cell invasion and tumor growth in GBM flank models, making this small molecule an ideal candidate for further development. We hypothesize that activating ULK1-modulated autophagy will be a therapeutic strategy for GBM growth inhibition

STEM-07. THE ATYPICAL METALLOPROTEINASE ADAMDEC1 MAINTAINS A NOVEL FGF MEDIATED SIGNALLING NETWORK IN GLIOBLASTOMA CANCER STEM CELLS

Abstract Glioblastomas (GBM) are lethal brain tumors where poor outcome is attributed to cellular heterogeneity, therapeutic resistance, and a highly infiltrative nature. These characteristics are preferentially linked to GBM cancer stem cells (CSCs), but how CSCs maintain their stemness is incompletely understood and the subject of intense investigation. CSCs rely both on cell intrinsic programs and microenvironmental interactions to regulate their maintenance. Recent work has demonstrated that CSCs can modulate their surrounding microenvironment by metalloproteinase expression, allowing direct modification of the extracellular matrix that provides structural tissue integrity, and instructive signalling via engagement of key cell surface receptors and sequestration of essential growth factors. Here, we identify a novel growth factor signalling loop that induces and maintains CSCs via an atypical metalloproteinase, a disintegrin and metalloproteinase domain-like protein decysin 1 (ADAMDEC1), secreted by CSCs. ADAMDEC1 solubilizes fibroblast growth factor-2 (FGF2) in the tumor microenvironment. We find that CSCs exclusively express FGF receptor 1 (FGFR1), which upon binding of FGF2 induces upregulation of Zinc finger E-box-binding homeobox 1 (ZEB1). ZEB1 is a regulator of stemness and tumor initiation, and therefore ADAMDEC1-FGF2-FGFR1 signalling promotes malignancy in GBM. We further show that ERK phosphorylation drives ZEB1, which regulates ADAMDEC1 expression, creating a positive feedback loop via repression of microRNA-203. Genetic or pharmacological targeting of components of this axis attenuates self-renewal and tumor growth. These findings reveal a new signalling axis for CSC maintenance and highlight ADAMDEC1 and FGFR1 as potential therapeutic targets in GBM. In addition, these findings provide a new paradigm for GBM growth through which key growth factors embedded within the extracellular matrix can be selectively accessed by CSC for their maintenance.

Molecular profiling of long-term IDH-wildtype glioblastoma survivors

Glioblastoma (GBM) represents an aggressive cancer type with a median survival of only 14 months. With fewer than 5% of patients surviving 5 years, comprehensive profiling of these rare patients could elucidate prognostic biomarkers that may confer better patient outcomes. We utilized multiple molecular approaches to characterize the largest patient cohort of isocitrate dehydrogenase (IDH)-wildtype GBM long-term survivors (LTS) to date. Retrospective analysis was performed on 49 archived formalin-fixed paraffin embedded tumor specimens from patients diagnosed with GBM at the Mayo Clinic between December 1995 and September 2013. These patient samples were subdivided into 2 groups based on survival (12 LTS, 37 short-term survivors [STS]) and subsequently examined by mutation sequencing, copy number analysis, methylation profiling, and gene expression. Of the 49 patients analyzed in this study, LTS were younger at diagnosis (P = 0.016), more likely to be female (P = 0.048), and MGMT promoter methylated (UniD, P = 0.01). IDH-wildtype STS and LTS demonstrated classic GBM mutations and copy number changes. Pathway analysis of differentially expressed genes showed LTS enrichment for sphingomyelin metabolism, which has been linked to decreased GBM growth, invasion, and angiogenesis. STS were enriched for DNA repair and cell cycle control networks. While our findings largely report remarkable similarity between these LTS and more typical STS, unique attributes were observed in regard to altered gene expression and pathway enrichment. These attributes may be valuable prognostic markers and are worth further examination. Importantly, this study also underscores the limitations of existing biomarkers and classification methods in predicting patient prognosis.

Abstract 2608: EGF signaling activates a TAZ-driven oncogenic program in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most aggressive and lethal primary brain tumor in humans. Hyperactivation of the signaling pathway driven by the epidermal growth factor (EGF) and its receptor (EGFR) are commonly occurring in GBM. The oncogenic transcriptional program driven by EGF signaling is still incompletely understood in GBM. Here, we found that EGF induces the upregulation and nuclear translocation of TAZ, an oncogenic transcriptional co-activator. TAZ and its paralog gene YAP are commonly known as downstream mediators of the Hippo signaling pathway. TAZ has been found as a driver of mesenchymal transition in GBM cells and promotes GBM radioresistance. Our results support that EGF induces TAZ transcription and the activation of TAZ gene targets, through the signaling axis including EGFR, STAT3 and ERK. EGFRvIII is the most frequently occurring EGFR mutation in GBM causing constitutively active EGFR. We found that EGFRvIII causes EGF-independent activation of TAZ and its downstream oncogenic targets. Next, we used next-generation sequencing to establish a genome-wide map of TAZ gene targets in GBM cells after EGF stimulation. The results suggest that EGF signaling activates a TAZ-driven oncogenic program including autocrine and paracrine factors that promote GBM growth, angiogenesis, stemness and immunoescape. Moreover, we found that TAZ targeting by shRNAs or the FDA-approved drug Verteporfin effectively inhibits the TAZ-driven oncogenic program activated by EGF stimulation or the EGFRvIII mutant. TAZ targeting also inhibits GBM cell growth and xenograft formation. In contrast, enforced TAZ induces the TAZ-driven oncogenic program and promotes GBM cell growth and xenograft formation. In conclusion, our results reveal a novel TAZ-driven oncogenic program that is activated by EGF signaling in GBM. Our discoveries strongly support TAZ as a therapeutic target for GBM and likely other human cancer with hyperactivation of EGF signaling. Our preclinical studies also provide a clinically translatable strategy for GBM therapy based on using the FDA-approved drug Verteporfin. Citation Format: Minling Gao, Yi Fu, Laterra John, Mingyao Ying. EGF signaling activates a TAZ-driven oncogenic program in glioblastoma [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 2608.

The kinase inhibitor SI113 induces autophagy and synergizes with quinacrine in hindering the growth of human glioblastoma multiforme cells

BackgroundGlioblastoma multiforme (GBM), due to its location, aggressiveness, heterogeneity and infiltrative growth, is characterized by an exceptionally dismal clinical outcome. The small molecule SI113, recently identified as a SGK1 inhibitor, has proven to be effective in restraining GBM growth in vitro and in vivo, showing also encouraging results when employed in combination with other antineoplastic drugs or radiotherapy. Our aim was to explore the pharmacological features of SI113 in GBM cells in order to elucidate the pivotal molecular pathways affected by the drug. Such knowledge would be of invaluable help in conceiving a rational offensive toward GBM.MethodsWe employed GBM cell lines, either established or primary (neurospheres), and used a Reverse-Phase Protein Arrays (RPPA) platform to assess the effect of SI113 upon 114 protein factors whose post-translational modifications are associated with activation or repression of specific signal transduction cascades.ResultsSI113 strongly affected the PI3K/mTOR pathway, evoking a pro-survival autophagic response in neurospheres. These results suggested the use of SI113 coupled, for maximum efficiency, with autophagy inhibitors. Indeed, the association of SI113 with an autophagy inhibitor, the antimalarial drug quinacrine, induced a strong synergistic effect in inhibiting GBM growth properties in all the cells tested, including neurospheres.ConclusionsRPPA clearly identified the molecular pathways influenced by SI113 in GBM cells, highlighting their vulnerability when the drug was administered in association with autophagy inhibitors, providing a strong molecular rationale for testing SI113 in clinical trials in associative GBM therapy.

Genome-Wide CRISPR-Cas9 Screens Expose Genetic Vulnerabilities and Mechanisms of Temozolomide Sensitivity in Glioblastoma Stem Cells

Glioblastoma therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells (GSCs) to interrogate function of the coding genome, we identify actionable pathways responsible for growth, which reveal the gene-essential circuitry of GBM stemness and proliferation. In particular, we characterize members of the SOX transcription factor family, SOCS3, USP8, and DOT1L, and protein ufmylation as important for GSC growth. Additionally, we reveal mechanisms of temozolomide resistance that could lead to combination strategies. By reaching beyond static genome analysis of bulk tumors, with a genome-wide functional approach, we reveal genetic dependencies within a broad range of biological processes to provide increased understanding of GBM growth and treatment resistance.

3131 ONCOSTREAMS: NOVEL DYNAMICS PATHOLOGICAL MULTICELLULAR STRUCTURES INVOLVED IN GLIOBLATOMA GROWTH AND INVASION

OBJECTIVES/SPECIFIC AIMS: Oncostreams represent a novel growth pattern of GBM. In this study we uncovered the cellular and molecular mechanism that regulates the oncostreams function in GBM growth and invasion. METHODS/STUDY POPULATION: We studied oncostreams organization and function using genetically engineered mouse gliomas models (GEMM), mouse primary patient derived GBM model and human glioma biopsies. We evaluated the molecular landscape of oncostreams by laser capture microdissection (LCM) followed by RNA-Sequencing and bioinformatics analysis. RESULTS/ANTICIPATED RESULTS: Oncostreams are multicellular structures of 10-20 cells wide and 2-400 μm long. They are distributed throughout the tumors in mouse and human GBM. Oncostreams are heterogeneous structures positive for GFAP, Nestin, Olig2 and Iba1 cells and negative for Neurofilament. Using GEMM we found a negative correlation between oncostream density and animal survival. Moreover, examination of patient’s glioma biopsies evidenced that oncostreams are present in high grade but no in low grade gliomas. This suggests that oncostreams may play a role in tumor malignancy. Our data also indicated that oncostreams aid local invasion of normal brain. Transcriptome analysis of oncostreams revealed 43 differentially expressed (DE) genes. Functional enrichment analysis of DE genes showed that “collagen catabolic processes”, “positive regulation of cell migration”, and “extracellular matrix organization” were the most over-represented GO biological process. Network analysis indicated that Col1a1, ACTA2, MMP9 and MMP10 are primary target genes. These genes were also overexpressed in more malignant tumors (WT-IDH) compared to the less malignant (IDH1- R132H) tumors. Confocal time lapse imagining of 3D tumor slices demonstrated that oncostreams display a collective motion pattern within gliomas that has not been seen before. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, oncostreams are anatomically and molecularly distinctive, regulate glioma growth and invasion, display collective motion and are regulated by the extracellular matrix. We propose oncostreams as novel pathological markers valuable for diagnosis, prognosis and designing therapeutics for GBM patients.

Abstract A045: Chlorotoxin redirects T-cells for specific and effective targeting against glioblastomas

Abstract Glioblastoma (GBM) is the most common type of primary brain tumor, with the standard therapy only modestly improving the prognosis, highlighting the necessity to develop advanced treatments. We and others have established the platform to potentiate immune response against GBMs using chimeric antigen receptor (CAR) engineered T-cells. Specifically, we have shown that intracranial administration of CAR T-cells can be well tolerated in patients with recurrent GBMs, together with some early clinical evidence of antitumor response. However, CAR T-cell therapy against GBMs is complicated by the inter- and intratumoral heterogeneity, while the single-targeting therapies only respond to a subset of tumor cells. The development of new CAR therapy would thus aim for targeting a wider range of tumor cells and bypassing antigen escape. Here, we took an approach different from conventional strategies of tumor antigen discovery, exploiting the tumor-binding potential of a natural peptide to develop CAR T-cells that broadly target GBMs. Chlorotoxin (CLTX) is a 36-amino acid peptide with demonstrated GBM-binding capability. Inspired by the utilization of CLTX in GBM tumor imaging, we used a fluorescence-conjugated CLTX to screen the freshly-dispersed primary GBM cells and patient-derived GBM neurospheres, and found that CLTX binding was more homogeneous than the expression of other GBM-associated antigens including EGFR, HER2 and IL13Rα2. Although CLTX has limited inhibitory effect on GBM growth, its broad binding to GBM cells illustrates the potential to be conjugated with a cytotoxic agent. Therefore, we generated CAR T-cells bearing CLTX as the antigen targeting domain. CLTX-CAR T-cells were able to get activated after stimulating with GBM cells, as indicated by their degranulation, cytokine production and immuno-synapse formation. Modification of CAR constructs revealed that CLTX-CAR T-cells with CD28 costimulatory signal exhibited potent effector activity, while the 4-1BB costimulation resulted in inadequate CAR activation. In both in vitro and in vivo models, CLTX-CAR T-cells effectively eliminated GBM cells and tumors, including the ones with no/low expression of EGFR, HER2 and IL13Rα2. Importantly, CLTX peptide exhibited negligible binding to a panel of normal cells from neural and other tissues, and CLTX-CAR T-cells showed no off-target effect against normal organs in tumor-bearing mouse models. Screening on patient-derived GBM neurospheres, we discovered that the expression of metalloproteinase (MMP)-2 on targeT-cells was correlated with the effector function of CLTX-CAR T-cells. Further, the antitumor function of CLTX-CAR T-cells was severely diminished against GBMs with MMP-2 knockdown. Consistent with the cytotoxicity of CLTX-CAR T-cells, MMP-2 expression was also present in a subgroup of GBM cells with undetectable levels of EGFR, HER2 and IL13Rα2 expression. Our results demonstrate for the first time that a peptide toxin can be successfully used as the tumor targeting domain of a CAR, which eliminates GBMs with high efficiency and selectivity. The CLTX-CAR has the potential to limit GBM heterogeneity and compensate current CAR T-cell therapies against solid tumors. Citation Format: Dongrui Wang, Vanessa Jonsson, Sarah Wright, Wen-Chung Chang, Xin Yang, Renate Starr, Alfonso Brito, Brenda Aguilar, Aniee Sarkissian, Lihong Weng, Stephen J Forman, Michael E Barish, Christine E. Brown. Chlorotoxin redirects T-cells for specific and effective targeting against glioblastomas [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A045.

Abstract 5828: Withaferin A and tumor treating fields synergistically inhibit glioma proliferation

Abstract Glioblastoma multiforme (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5-10%). Current interventions consist of equally aggressive surgical resection, radiotherapy, and chemotherapy. However, this cancer is heterogeneous and not easily accessible so standardized, non-specific therapies are often ineffective. Novel, unorthodox approaches against GBM need consideration. Withaferin A is a steroidal lactone that originates from the roots and leaves of the winter cherry plant (Withania somnifera). Within Ayurvedic medicine, Withania somnifera extracts are applied to many disorders including several cancers. Extracts such as AshwaMAX can contain up to 4.3% (w/w) Withaferin A, a biologically active compound from Withania somnifera. Tumor Treating Fields (TTFields) are thought to disrupt mitotic spindle formation and stymie proliferation of actively dividing cancer cells. We report on the anti-cancer properties of both Withaferin A and TTFields. We also examined the hypothesis that combining TTFields with Withaferin A would synergistically inhibit GBM growth. We used GBM cells (U87-MG, GBM2, and GBM39) and breast adenocarcinoma cells (MDA-MB-231) isolated from human primary tumors. Each GBM cell line was modified to express firefly luciferase. Proliferative potential was assessed by bioluminescent imaging (BLI), cell counting via hemocytometer, or through the CellTiter-Blue® viability assay. Intracranial orthotopic U87-MG GBM xenografts were grown in the right frontal lobe of female nude mice (n=5/experiment). TTFields were imposed on cell cultures with the inovitroTM system from Novocure Ltd. Neurosphere cultures (U87-MG, GBM2, and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM (Withaferin A equivalent), respectively, and by Withaferin A with IC50 of 0.31, 0.28, and 0.25 µM, respectively. Oral gavage, every other day, of AshwaMAX (40mg/kg per dose) significantly reduced BLI signal (n=5 mice/group, p<0.02, four-parameter non-linear regression analysis) in preclinical models. Comsol modeling showed that the inovitroTM device imposed optimal TTFields in the center of the cell culture dishes. TTFields at 4 V/cm significantly inhibited (n=4 measurements per time point, p<0.001, 2-way ANOVA) growth of MDA-MB-231 cancer lines and all three human GBM lines. The effects were due to the TTFields alone and not to the slight temperature rises caused by the TTFields. Combination of Withaferin A (10-100 nM) with TTFields (4 V/cm) significantly inhibited GBM growth, to a degree beyond that of Withaferin A or TTFields alone. Poisson-based analysis of significance for regression indicated that the interaction of the combined treatments on GBM cells was synergistic (p<0.01, n=3 experiments). The findings suggest a novel approach to treat GBM in a manner that is likely better than either treatment (Withaferin A or TTFields) alone and that is synergistic. Citation Format: Edwin Chang, Chirag Patel, Christoph Pohling, Nooshin Beygui, Dong Ho Ha, Sanjiiv S. Gambhir. Withaferin A and tumor treating fields synergistically inhibit glioma proliferation [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 5828.

Abstract 3342: Truncated glioma-associated oncogene homolog 1 (tGLI1) is a novel mediator of mesenchymal subtype of glioblastoma and a novel transcriptional activator of CD44

Abstract The subpopulation of stem-like cells, or glioma stem cells (GSCs), is a key driver of tumor initiation, recurrence, and chemoresistance. Recently, patient-derived GSC lines were identified to contain two mutually exclusive subtypes termed proneural and mesenchymal. Mesenchymal GBM and GSCs were found to be more aggressive and radioresistant; however, the molecular pathways supporting mesenchymal GBM and mesenchymal GSCs are not well understood. To address this knowledge gap, we examined truncated glioma-associated oncogene homolog 1 (tGLI1) for its role in mesenchymal subtypes of GBM and GSCs. Discovered in our laboratory, tGLI1 is a novel alternatively spliced, gain-of-function variant of GLI1 zinc-finger transcription factor with an in-frame deletion of 41 codons retains all of the known functional domains of GLI1, but gains the ability to activate genes not regulated by GLI1. In this study, we report for the first time that tGLI1 is a tumor-specific transcription factor that facilitates GBM growth, enriches in the mesenchymal subtype of GBM and GSCs, and promotes mesenchymal GSCs via transcriptionally activating CD44 expression. Using an orthotopic GBM xenograft mouse model, we observed that tGLI1-overexpressing tumors grew more aggressively, with increased proliferation and angiogenesis, compared to control and GLI1-overexpressing xenografts. IHC analysis using tGLI1- and GLI1-selective antibodies we developed showed that tGLI1 was significantly expressed in GBM specimens but undetectable in normal brains whereas GLI1 was expressed in both tissues. Datamining further showed that tGLI1 activation signature (tGAS) was correlated with glioma grade, tumor angiogenesis, and poor overall survival, and that GBMs with high tGAS were enriched with mesenchymal GBM and GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared to the monolayer culture. Mesenchymal GSCs expressed more tGLI1 than proneural GSCs. Ectopic tGLI1 expression significantly enhanced the ability of GSCs to yield neurospheres in vitro and form aggressive tumors in mouse brains. Finally, we examined the ability of tGLI1 to regulate stemness genes and found that tGLI1 binds to and transactivates the promoter of the CD44 gene, a marker and mediator for mesenchymal GSCs. Collectively, these findings advanced our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSCs. Citation Format: Tadas K. Rimkus. Truncated glioma-associated oncogene homolog 1 (tGLI1) is a novel mediator of mesenchymal subtype of glioblastoma and a novel transcriptional activator of CD44 [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 3342.

Reactive species balance via GTP cyclohydrolase I regulates glioblastoma growth and tumor initiating cell maintenance.

Depending on the level, differentiation state, and tumor stage, reactive nitrogen and oxygen species inhibit or increase cancer growth and tumor initiating cell maintenance. The rate-limiting enzyme in a pathway that can regulate reactive species production but has not been thoroughly investigated in glioblastoma (GBM; grade IV astrocytoma) is guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1). We sought to define the role of GCH1 in the regulation of GBM growth and brain tumor initiating cell (BTIC) maintenance. We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples, and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production, and survival in orthotopic patient-derived xenograft models were determined. GCH1 was expressed in GBMs with elevated but not exclusive RNA and protein levels in BTICs in comparison to non-BTICs. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In converse experiments, GCH1 knockdown with short hairpin RNA led to GBM cell growth inhibition and reduced self-renewal in association with decreased CD44 expression. GCH1 was critical for controlling reactive species balance, including suppressing reactive oxygen species production, which mediated GCH1 cell growth effects. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence, and worse survival. GCH1 expression in established GBMs is pro-tumorigenic, causing increased growth due, in part, to promotion of BTIC maintenance and suppression of reactive oxygen species.

Phostine PST3.1a Targets MGAT5 and Inhibits Glioblastoma-Initiating Cell Invasiveness and Proliferation.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and accounts for a significant proportion of all primary brain tumors. Median survival after treatment is around 15 months. Remodeling of N-glycans by the N-acetylglucosamine glycosyltransferase (MGAT5) regulates tumoral development. Here, perturbation of MGAT5 enzymatic activity by the small-molecule inhibitor 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST3.1a) restrains GBM growth. In cell-based assays, it is demonstrated that PST3.1a alters the β1,6-GlcNAc N-glycans of GBM-initiating cells (GIC) by inhibiting MGAT5 enzymatic activity, resulting in the inhibition of TGFβR and FAK signaling associated with doublecortin (DCX) upregulation and increase oligodendrocyte lineage transcription factor 2 (OLIG2) expression. PST3.1a thus affects microtubule and microfilament integrity of GBM stem cells, leading to the inhibition of GIC proliferation, migration, invasiveness, and clonogenic capacities. Orthotopic graft models of GIC revealed that PST3.1a treatment leads to a drastic reduction of invasive and proliferative capacity and to an increase in overall survival relative to standard temozolomide therapy. Finally, bioinformatics analyses exposed that PST3.1a cytotoxic activity is positively correlated with the expression of genes of the epithelial-mesenchymal transition (EMT), while the expression of mitochondrial genes correlated negatively with cell sensitivity to the compound. These data demonstrate the relevance of targeting MGAT5, with a novel anti-invasive chemotherapy, to limit glioblastoma stem cell invasion. Mol Cancer Res; 15(10); 1376-87. ©2017 AACR.

Abstract 3968: Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth

Abstract Glioblastoma (GBM) development and therapeutic resistance has been accompanying with the tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). TAMs are heterogeneous cell populations of immune regulatory myeloid-derived suppressor cells (MDSCs) and polarization of anti-tumor macrophages (M1) into pro-tumor macrophages (M2). We investigated the role of myeloid cell NF-κB signaling in orthotopic GBM model using immune deficient and immune competent hosts. Interestingly, conditional deletion of canonical NF-κB signaling (p65) with Lysm-Cre (p65 KO) in myeloid cells, significantly inhibited syngeneic GL261 tumor growth in immune-competent mice compared to control mice. We studied the TAMs recruitment to the tumor and their polarization under the influence of TME. P65 KO mice displayed decreasing trend of immune cell infiltration (CD45), which phenotyped as decreased F4/80+, CD68+, CD206+ (M2) and Gr1+CD11b+ (MDSCs) macrophages, compared to control mice. This was associated with the increased CD80+ (M1) macrophages, increasing trend of CD4+ and CD8+ cytotoxic T cells, and decreased CD44+ mesenchymal cancer stem cells (CSCs) populations in the TME. Cytokine array data indicated that loss of canonical NF-κB signaling within the TAMs was implicated in increased production of IFNγ, IGF1, MCP1, MIP1α, and TNFα cytokines. Co-culture of T cells with p65 KO or control MDSCs identified increased proliferation of T cells with p65 KO MDSCs compared to control MDSCs. Conversely, GBM patient-derived xenografts and U251 GBM cell line-derived tumors showed increasing trend of growth in immune-deficient mice, following the transplantation of p65 KO bone marrow (BM) compared to control BM. Pro-tumor macrophages and CSCs were increased and T cell populations were decreased in human tumors grown in immune deficient mice transplanted with p65 KO BM, compared with control BM. In addition, analysis of human data set revealed higher expression of p65 subunit of NF-κB complex in brain tumor stroma compared to the tumor cells. The study suggests that canonical NF-κB signaling in TAMs is required for the tumor-promoting macrophage polarization and GBM growth in immunocompetent host compared to immune deficient host. Therefore, targeting myeloid-specific NFκB signaling in GBM could inhibit the immune suppressive TAMs and improve the anti-tumor immunity. Citation Format: Bhagelu R. Achyut, Jennifer Bradford, Kartik Angara, Mohammad Rashid, Meenu Jain, Thaiz Borin, ASM Iskander, Roxan Ara, Ali Arbab. Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth [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 3968. doi:10.1158/1538-7445.AM2017-3968

Abstract 2889: V-ATPase control of EV signaling in glioma stem cells

Abstract Background: Recent evidences highlighted that GBM secreted microvesicles (EVs), particularly exosomes (Exo) and large oncosomes (LO), play a major role in the cross-talk between tumor cell and non-neoplastic parenchyma. How GBMs manage to thrive in a highly unfavorable, acidic microenvironment is still unclear, but recent work from our group has identified the vacuolar pump H+-ATPase (V-ATPase) as an important effector of GBM growth and glioma stem cells (GSC) maintenance. Additionally, in ExoCarta database V-ATPase subunits have been described in Exo from different cancer cell types. Taken together, these data identify V-ATPase as an important driver of gliomagenesis, and a novel, actionable therapeutic target for disease intervention. However, the role of V-ATPase in reprogramming the GBM microenvironment has not been previously investigated. Methods: Exo and LO were isolated by an Invitrogen kit and serial centrifugation, respectively, from media of patients’ derived GBM neurospheres, enriched in GSC (NS, n=12) or differentiated cultures (n=8). For EVs internalization studies, Exo or LO were stained using FM 1-43 FX dye and the process was followed live for 30’ and at selected time points (30’-4h-24h), using a confocal microscopy or flow cytometry (FACS). Electron microscopy, FACS (of Exo stained with CellTrace and SytoRNA in combination with CD63 coated beads), Nanosight and immunoblotting (for CD63, CD9 and Clathrin) analyses were used to confirm EVs subtypes. Cultures from patients’ derived brain tumor margins or primary GBM (differentiated and not) were used as EVs-recipient cells. miRNA profiling was performed using Taqman Low density arrays and analyzed by R packages. Gene Ontology analysis was performed by DAVID. The study was approved by the Institutional Ethical Committee. Results: NS are able to produce different EVs, which are internalized by recipient cells after 4 and up to 24 hours of co-culture. Both Exo than LO from NS are able to significantly increase cell growth in recipient cells (brain tumor margins and primary GMB differentiated monolayers), and this effect is stronger with EVs produced by NS with higher V-ATPase expression (V-ATPaseHIGH NS). Primary GBM cells after co-culture with EVs are able to produce a higher number of NS and V-ATPase activity block by BafilomycinA1 in NS-producing EVs completely revert this effect. Finally, the co-culture of V-ATPaseLOW NS with EVs from V-ATPaseHIGH NS increases their motility in collagen matrixes. At molecular level, profiling of Exo-derived miRNAs distinguishes differentiated cultures from NS and, among NS, V-ATPaseHIGH cultures. In silico analysis and annotation of miRNA target genes from V-ATPaseHIGH–derived Exo showed an enrichment of cancer, cell cycle and PI3K/Akt pathways. Conclusions: Altogether, these data point toward the central role of different EV types in GBM communication and suggest a role of the V-ATPase proton pump in regulating EV’s contents. Citation Format: Irene Bertolini, Andrea Terrasi, Andrea Di Cristofori, Silvano Bosari, Valentina Vaira. V-ATPase control of EV signaling in glioma stem cells [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 2889. doi:10.1158/1538-7445.AM2017-2889

Fibronectin-adherent peripheral blood derived mononuclear cells as Paclitaxel carriers for glioblastoma treatment: An in vitro study

BackgroundGlioblastoma (GBM) represents the most aggressive malignant brain tumor in adults, with a risible median life expectancy despite gold standard treatment. Novel drug-delivery methods have been explored. Here we evaluated the possibility to use mononuclear cells (MCs) belonging to the monocytic-dendritic lineage as drug-carrier. MethodsMCs were obtained from 10 patients harboring a GBM, and from healthy volunteers, considered as controls. GBM tissue was also obtained from patients. MCs were cultured and the adherent population on fibronectin (FN-MCs), after immunocytochemistry and flow cytometry characterization, was loaded with Paclitaxel (FN-MCs-PTX). Antiproliferative and migration activity of FN-MCs-PTX was evaluated in two-dimensional (2D) and three-dimensional (3D) co-culture assays with red fluorescent U87 Malignant Glioma cells and primary GBM cells. Antiangiogenic properties of FN-MCs-PTX were tested on cultures with endothelial cells. ResultsPhenotypical characterization showed a high expression of monocytic-dendritic markers in GBM cells and FN-MCs. FN-MCs demonstrated to effectively uptake PTX and to strongly inhibit GBM growth in vitro (P < 0.01). Moreover, tumor-induced migration of MCs, although partially affected by the PTX cargo, remained statistically significant when compared with unprimed cells and this was confirmed in a 3D Matrigel model (P < 0.01) and in a Trans-well assay (P < 0.01). FN-MCs-PTX also disclosed considerable antiangiogenic properties. DiscussionOur results suggest that the fibronectin-adherent population of MCs isolated from peripheral blood can be an effective tool to inhibit GBM growth. Given the relative facility to obtain such cells and the short time needed for their culture and drug loading this approach may have potential as an adjuvant therapy for GBM.

Diffusion Tensor (DTI) Magnetic Resonance Imaging–Based Simulations of GBM Growth and Response to Radiation Therapy

Diffusion Tensor (DTI) Magnetic Resonance Imaging–Based Simulations of GBM Growth and Response to Radiation Therapy