Glioblastoma Multiforme Patients Research Articles

Identification of TNFAIP6 as a hub gene associated with the progression of glioblastoma by weighted gene co-expression network analysis.

This study aims to discover the genetic modules that distinguish glioblastoma multiforme (GBM) from low‐grade glioma (LGG) and identify hub genes. A co‐expression network is constructed using the expression profiles of 28 GBM and LGG patients from the Gene Expression Omnibus database. The authors performed gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG) analysis on these genes. The maximal clique centrality method was used to identify hub genes. Online tools were employed to confirm the link between hub gene expression and overall patient survival rate. The top 5000 genes with major variance were classified into 18 co‐expression gene modules. GO analysis indicated that abnormal changes in ‘cell migration’ and ‘collagen metabolic process’ were involved in the development of GBM. KEGG analysis suggested that ‘focal adhesion’ and ‘p53 signalling pathway’ regulate the tumour progression. TNFAIP6 was identified as a hub gene, and the expression of TNFAIP6 was increased with the elevation of pathological grade. Survival analysis indicated that the higher the expression of TNFAIP6, the shorter the survival time of patients. The authors identified TNFAIP6 as the hub gene in the progression of GBM, and its high expression indicates the poor prognosis of the patients.

Expression and Prognostic Role of Glia Maturation Factor-γ in Gliomas.

BackgroundGlia maturation factor-γ (GMFG) regulates actin cytoskeletal organization and promotes the invasion of cancer cells. However, its expression pattern and molecular function in gliomas have not been clearly defined.MethodsIn this study, public datasets comprising 2,518 gliomas samples were used to explore GMFG expression and its correlation with malignancy in gliomas. Immunohistochemistry (IHC) staining was performed to determine the expression of GMFG in gliomas using an in-house cohort that contained 120 gliomas samples. Gene ontology enrichment analysis was conducted using the DAVID tool. The correlation between GMFG expression and immune cell infiltration was evaluated using TIMER, Tumor Immune Single-Cell Hub (TISCH) database, and IHC staining assays. The Kaplan–Meier analysis was performed to determine the prognostic role of GMFG and its association with temozolomide (TMZ) response in gliomas.ResultsThe GMFG expression was higher in gliomas compared with non-tumor brain tissues both in public datasets and in-house cohort. High expression of GMFG was significantly associated with WHO grade IV, IDH 1/2 wild-type, and mesenchymal (ME) subtypes. Bioinformatic prediction and IHC analysis revealed that GMFG expression obviously correlated with the macrophage marker CD163 in gliomas. Moreover, both lower grade glioma (LGG) and glioblastoma multiforme (GBM) patients with high GMFG expression had shorter overall survival than those with low GMFG expression. These results indicate that GMFG may be a therapeutic target for the treatment of such patients. Patients with low GMFG expression who received chemotherapy had a longer survival time than those with high GMFG expression. For patients who received ion radiotherapy (IR) only, the GMFG expression level had no effect on the overall survival neither in CGGA and TCGA datasets.ConclusionThe GMFG is a novel prognostic biomarker for patients with both LGG and GBM. Increased GMFG expression is associated with tumor-associated macrophages (TAMs) infiltration and with a bad response to TMZ treatment.

Morusin Enhances Temozolomide Efficiency in GBM by Inducing Cytoplasmic Vacuolization and Endoplasmic Reticulum Stress.

Glioblastoma multiforme (GBM) is an aggressive brain tumor with high risks of recurrence and mortality. Chemoradiotherapy resistance has been considered a major factor contributing to the extremely poor prognosis of GBM patients. Therefore, there is an urgent need to develop highly effective therapeutic agents. Here, we demonstrate the anti-tumor effect of morusin, a typical prenylated flavonoid, in GBM through in vivo and in vitro models. Morusin showed selective cytotoxicity toward GBM cell lines without harming normal human astrocytes when the concentration was less than 20 µM. Morusin treatment significantly induced apoptosis of GBM cells, accompanied by the activation of endoplasmic reticulum (ER) stress, and the appearance of cytoplasmic vacuolation and autophagosomes in cells. Then, we found the ER stress activation and cytotoxicity of morusin were rescued by ER stress inhibitor 4-PBA. Furthermore, morusin arrested cell cycle at the G1 phase and inhibited cell proliferation of GBM cells through the Akt–mTOR–p70S6K pathway. Dysregulation of ERs and cell cycle in morusin exposed GBM cells were confirmed by RNA-seq analysis. Finally, we demonstrated the combination of morusin and TMZ remarkably enhanced ER stress and displayed a synergistic effect in GBM cells, and suppressed tumor progression in an orthotopic xenograft model. In conclusion, these findings reveal the toxicity of morusin to GBM cells and its ability to enhance drug sensitivity to TMZ, suggesting the potential application value of morusin in the development of therapeutic strategies for human GBM.

Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion

High-grade gliomas are the most fatal brain tumors. Grade 4 gliomas are called glioblastoma multiforme (GBM), which are associated with the poorest survival and a 5-year survival rate of less than 4%. Many patients with GBM developed concomitant cognitive dysfunctions and epilepsy. Although the cognitive decline is well defined in glioblastomas, the neurotoxic factors underlying this pathology are not well understood in GBM patients. In this study, we aimed to investigate whether GBM-derived exosomes play a role in neuronal toxicity. For this purpose, exosomes obtained from T98G and U373 GBM cells were applied to primary neuron culture at different concentrations. Subsequently, MTT, LDH, GSH, TAS, and TOS tests were performed. Both GBM-derived exosomes induced a dose-dependent and statistically significant increase of LDH release in cerebellar neurons. MTT assay revealed as both T98G and U373 GBM-derived exosomes induced dose-dependent neurotoxic effects in cerebellar neurons. To the best of our knowledge, this study is the first study demonstrating the toxic potential of GBM-derived exosomes to primary neurons, which may explain the peritumoral edema and cognitive decline in GBM patients.

Identification of a prognostic microenvironment-related gene signature in glioblastoma multiforme patients treated with carmustine wafers.

Identification of a prognostic microenvironment-related gene signature in glioblastoma multiforme patients treated with carmustine wafers.

An optimized ionizable cationic lipid for brain tumor-targeted siRNA delivery and glioblastoma immunotherapy

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a high mortality rate. Immunotherapy has achieved promising clinical results in multiple cancers, but shows unsatisfactory outcome in GBM patients, and poor drug delivery across the blood-brain barrier (BBB) is believed to be one of the main limitations that hinder the therapeutic efficacy of drugs. Herein, a new cationic lipid nanoparticle (LNP) that can efficiently deliver siRNA across BBB and target mouse brain is prepared for modulating the tumor microenvironment for GBM immunotherapy. By designing and screening cationic LNPs with different ionizable amine headgroups, a lipid (named as BAMPA-O16B) is identified with an optimal acid dissociation constant (pKa) that significantly enhances the cellular uptake and endosomal escape of siRNA lipoplex in mouse GBM cells. Importantly, BAMPA-O16B/siRNA lipoplex is highly effective to deliver siRNA against CD47 and PD-L1 across the BBB into cranial GBM in mice, and downregulate target gene expression in the tumor, resulting in synergistically activating a T cell-dependent antitumor immunity in orthotopic GBM. Collectively, this study offers an effective strategy for brain targeted siRNA delivery and gene silencing by optimizing the physicochemical property of LNPs. The effectiveness of modulating immune environment of GBM could further be expanded for potential treatment of other brain tumors.

Characterization of Different Subtypes of Immune Cell Infiltration in Glioblastoma to Aid Immunotherapy.

Glioblastoma multiforme (GBM) has been identified as a frequently occurring adult primary brain cancer that is highly aggressive. Currently, the prognostic outcome for GBM patients is dismal, even with intensive treatment, and the median overall survival (OS) is 14.6 months. Immunotherapy, which is specific at the cellular level and can generate persistent immunosurveillance, is now becoming a promising tool to treat diverse cancers. However, the complicated nature of the tumor microenvironment (TME) makes it challenging to develop anti-GBM immunotherapy because several cell types, cytokines, and signaling pathways are involved in generating the immunosuppressive environment. Novel immunotherapies can illustrate novel tumor-induced immunosuppressive mechanisms. Here, we used unsupervised clustering analysis to identify different subtypes of immune cell infiltration that actuated different prognoses, biological actions, and immunotherapy responses. Gene cluster A, with a hot immune cell infiltration phenotype, had high levels of immune-related genes (IRGs), which were associated with immune pathways including the interferon-gamma response and interferon-alpha response, and had low IDH1 and ATRX mutation frequencies. Gene cluster B, a cold immune cell infiltration subtype, exhibited a high expression of the KCNIP2, SCRT1, CPLX2, JPH3, UNC13A, GABRB3, ARPP21, DLGAP1, NRXN1, DLL3, CA10, MAP2, SEZ6L, GRIA2, and GRIA4 genes and a low expression of immune-related genes, i.e., low levels of immune reactivity. Our study highlighted the complex interplay between immune cell infiltration and genetic mutation in the establishment of the tumor immune phenotype. Gene cluster A was identified as an important subtype with a better prognosis and improved immunotherapy response.

C1R, CCL2, and TNFRSF1A Genes in Coronavirus Disease-COVID-19 Pathway Serve as Novel Molecular Biomarkers of GBM Prognosis and Immune Infiltration.

Glioblastoma multiforme (GBM) is a prevalent intracranial brain tumor associated with a high rate of recurrence and treatment difficulty. The prediction of novel molecular biomarkers through bioinformatics analysis may provide new clues into early detection and eventual treatment of GBM. Here, we used data from the GTEx and TCGA databases to identify 1923 differentially expressed genes (DEGs). GO and KEGG analyses indicated that DEGs were significantly enriched in immune response and coronavirus disease-COVID-19 pathways. Survival analyses revealed a significant correlation between high expression of C1R, CCL2, and TNFRSF1A in the coronavirus disease-COVID-19 pathway and the poor survival in GBM patients. Cell experiments indicated that the mRNA expression levels of C1R, CCL2, and TNFRSF1A in GBM cells were very high. Immune infiltration analysis revealed a significant difference in the proportion of immune cells in tumor and normal tissue, and the expression levels of C1R, CCL2, and TNFRSF1A were associated with immune cell infiltration of GBM. Additionally, the protein-protein interaction networks of C1R, CCL2, and TNFRSF1A involved a total of 65 nodes and 615 edges. These results suggest that C1R, CCL2, and TNFRSF1A may be used as molecular biomarkers of prognosis and immune infiltration in GBM patients in the future.

The immunosuppressive role of indoleamine 2, 3-dioxygenase in glioblastoma: mechanism of action and immunotherapeutic strategies.

Glioblastoma multiforme (GBM) is a fatal brain tumor in adults with a bleak diagnosis. Expansion of immunosuppressive and malignant CD4 + FoxP3 + GITR + regulatory T cells is one of the hallmarks of GBM. Importantly, most of the patients with GBM expresses the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). While IDO1 is generally not expressed at appreciable levels in the adult central nervous system, it is rapidly stimulated and highly expressed in response to ongoing immune surveillance in cancer. Increased levels of immune surveillance in cancer are thus related to higher intratumoral IDO expression levels and, as a result, a worse OS in GBM patients. Conversion of the important amino acid tryptophan into downstream catabolite known as kynurenines is the major function of IDO. Decreasing tryptophan and increasing the concentration of immunomodulatory tryptophan metabolites has been shown to induce T-cell apoptosis, increase immunosuppressive programming, and death of tumor antigen-presenting dendritic cells. This observation supported the immunotherapeutic strategy, and the targeted molecular therapy that suppresses IDO1 activity. We review the current understanding of the role of IDO1 in tumor immunological escape in brain tumors, the immunomodulatory effects of its primary catabolites, preclinical research targeting this enzymatic pathway, and various issues that need to be overcome to increase the prospective immunotherapeutic relevance in the treatment of GBM malignancy.

A 3-miRNA Signature Enables Risk Stratification in Glioblastoma Multiforme Patients with Different Clinical Outcomes.

Malignant gliomas constitute a complex disease phenotype that demands optimum decision-making as they are highly heterogeneous. Such inter-individual variability also renders optimum patient stratification extremely difficult. microRNA (hsa-miR-20a, hsa-miR-21, hsa-miR-21) expression levels were determined by RT-qPCR, upon FFPE tissue sample collection of glioblastoma multiforme patients (n = 37). In silico validation was then performed through discriminant analysis. Immunohistochemistry images from biopsy material were utilized by a hybrid deep learning system to further cross validate the distinctive capability of patient risk groups. Our standard-of-care treated patient cohort demonstrates no age- or sex- dependence. The expression values of the 3-miRNA signature between the low- (OS > 12 months) and high-risk (OS < 12 months) groups yield a p-value of <0.0001, enabling risk stratification. Risk stratification is validated by a. our random forest model that efficiently classifies (AUC = 97%) patients into two risk groups (low- vs. high-risk) by learning their 3-miRNA expression values, and b. our deep learning scheme, which recognizes those patterns that differentiate the images in question. Molecular-clinical correlations were drawn to classify low- (OS > 12 months) vs. high-risk (OS < 12 months) glioblastoma multiforme patients. Our 3-microRNA signature (hsa-miR-20a, hsa-miR-21, hsa-miR-10a) may further empower glioblastoma multiforme prognostic evaluation in clinical practice and enrich drug repurposing pipelines.

Abstract 2727: Overexpression of protocadherin related 15 (PCDH15) gene predicts better prognosis in low-grade glioma patients

Abstract Background: Gliomas are the most common tumors arising from the brain. According to the WHO classification, gliomas are divided into low- and high-grade gliomas based on their aggressiveness and degrees of differentiation. Protocadherin Related 15 (PCDH15) gene is a member of the cadherin superfamily that encode membrane proteins involved in cell-cell adhesion. In this study, we aimed to investigate the prognostic value of PCDH15 expression in Low-Grade Gliomas (LGG) and Glioblastoma Multiforme (GBM). Methods: We conducted a bioinformatics analysis for PCDH15 in LGG and GBM patients’ data using their respective datasets from the Cancer Genomic Atlas (TCGA). GEPIA and GENT2 web-based tools were used to confirm the differential expression of PCDH15 in LGG and GBM patients in comparison with normal brain tissue. We used the cBioPortal database to compare PCDH15 expression across molecular and histological glioma subtypes. Also, the SurvExpress tool was used to assess the prognostic value of PCDH15 on overall survival (OS). To further understand the underlying molecular biology of PCDH15’s function, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted for significantly co-expressed genes (|spearman correlation coefficient|≥ 0.5) with PCDH15 using the DAVID 6.8 bioinformatics tool. Results: LGG and GBM both had statistically significant differences in PCDH15 expression compared with normal brain tissue. PCDH15 expression was significantly higher in LGG patients compared with GBM patients (mean log2 (TPM + 1): 9.5 vs 5.6, P &amp;lt; 0.0001), with the highest expression in patients with mutated IDH genes. In the LGG cohort, higher PCDH15 expression was associated with longer OS (hazard ratio: 2.44, 95% confidence interval: 1.69 - 3.53, P = 1.949E-6) while no significant difference was found in the GBM cohort (hazard ratio: 1.07, 95% confidence interval: 0.74 - 1.53, P = 0.7304). GO analyses for co-expressed genes in LGG indicated a role in the biological process (BP) DNA-templated regulation of transcription with a cellular component (CC) in the nucleus and the molecular functions (MF) “histone demethylase activity”, “hydrolase activity, acting on acid anhydrides” and “zinc ion binding”. While in GBM, GO analysis showed enrichment in the BP terms, “neuron cell-cell adhesion”, “positive regulation of synapse assembly”, and the CC terms, “cell junction”, “axon”, “plasma membrane”. KEGG pathway analysis did not retrieve any significantly enriched pathways in both diseases. Conclusion: Our findings demonstrate a unique pattern of PCDH15 overexpression in low-grade gliomas which was translated into a substantial impact on patients’ survival. Functional enrichment analysis indicated transcription regulation as a potential mechanism for PCDH15 in LGG, further supporting PCDH15's potential as a therapeutic target. Citation Format: Hassan Mohammed Abushukair, Bayan Osama Abu Alragheb. Overexpression of protocadherin related 15 (PCDH15) gene predicts better prognosis in low-grade glioma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2727.

Abstract 6058: The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme

Abstract Purpose: Glioblastoma multiforme (GBM) remains a fatal disease despite aggressive treatment approaches. Post-surgical radiation therapy (RT) is the only treatment that significantly improves survival of GBM patients but recurrence is inevitable. Elucidating the mechanisms of resistance to RT could transform GBM patient outcomes. The purpose of this study was to elucidate the effect of radiation on the serine synthesis pathway (SSP) and the role of SSP in boosting antioxidant defenses in irradiated GBM cells and promoting radioresistance. Methods: We compared copy number alterations (CNA) and amplification of SSP enzymes (PHGDH, PSAT1, PSPH, SHMT1/2) in low-grade gliomas (LGG, n = 511) and GBM (n = 575) via TCGA pan-cancer analysis. In vitro HPLC/MS-based metabolomics assays were used to study the changes in overall metabolite levels induced by radiation in patient-derived GBM specimens. Targeted metabolomics with 13C-labeled glucose was used for tracking the fate of glucose towards SSP intermediates and antioxidant species. Gene expression levels after radiation were measured via molecular biology approaches. De novo SSP was targeted via pharmacological inhibition of PHGDH. Modified clonogenic, sphere forming assays (SFA) on gliomasphere cultures were used to determine radiosensitivity. Dependence on extracellular serine/glycine was determined via serine/glycine depletion experiments. Results: The TCGA analysis revealed increased CNAs in all the de novo SSP enzymes in GBM tumors relative to the LGGs. 82% of GBM tumors had CNAs or amplification of the PSPH gene, which codes for the ultimate enzyme in serine synthesis. In vitro metabolomics assays revealed that radiation increases the levels of serine, glycine and cystine in gliomaspheres, and this is accompanied by elevated levels of reduced glutathione (GSH). Targeted glucose metabolomics revealed radiation-induced upregulation of the de novoSSP, with a concomitant increase in gene expression of SSP enzymes. Downstream of the SSP, metabolomics data show that irradiated GBM cells elevate nucleotide and precursor levels, likely to support DNA damage repair mechanisms after radiation. The inhibition of PHGDH, the first rate-limiting enzyme in de novo SSP, significantly reduced GBM cell survival in vitro. Extracellular serine/glycine appeared to also be important contributors to radiation survival. Gene expression of SLC1A4, an amino acid transporter, was upregulated by radiation, while depletion of extracellular serine/glycine led to significant decreased survival in in vitro SFAs. Conclusion: Here we provide evidence that the de novo serine synthesis pathway is an important contributor to the metabolic reprogramming of GBM cells after radiation that promotes radiation resistance. These data call for further investigations to evaluate the radiosensitizing potential of targeting de novo SSP in GBM. Citation Format: Justine Bailleul, Yangjingyi Ruan, Erina Vlashi. The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6058.

Abstract 5325: Image-based functional precision medicine for repurposing neuroactive drugs in glioblastoma

Abstract Treatment of glioblastoma multiforme (GBM), the most aggressive form of primary brain cancer, has essentially not advanced over the past few decades. Numerous challenges hinder the successful development of new therapies, including drug delivery across the blood-brain barrier (BBB), the complexity of the tumor microenvironment (TME), and the lack of clinically predictive cancer models. Here, we present the results of an image-based ex vivo drug-testing platform that addresses these therapeutic roadblocks. To demonstrate the clinical utility of our platform, in a retrospective cohort of 14 GBM patients, we show that ex vivo sensitivity to Temozolomide (TMZ, 1st-line GBM chemotherapy), is associated with longer progression free survival (PFS) and overall survival (OS). Next, by screening 150 clinically approved drugs across 27 GBM surgical patient samples, we identify a set of BBB-permeable neuroactive drugs with anti-glioma activity. These neurological drugs display remarkably consistent on-target killing of cancer cells with minimal toxicity to non-malignant TME cells across both primary and recurrent GBM samples. Single-cell transcriptional profiling of GBM patient samples and functional genetics reveals novel glioma-dependencies on neurological drug-target expression. Furthermore, a drug-target network enrichment analysis uncovers an AP1/BTG/TP53 gene signature associated with the anti-glioma activity of neurological drugs. In silico screening of over 1 million compounds for this common gene signature identified additional drug hits that could be validated in patient samples with 90% accuracy. Multiplexed transcriptomics revealed AP-1 transcription factor family activation to be the common underlying feature of neurological drugs with anti-glioma activity. Among the most promising candidate drugs, we identify the atypical antidepressant Vortioxetine as the strongest inducer of this gene signature, and confirm its efficacy in vivo across multiple mouse models. Vortioxetine in combination with Temozolomide or Lomustine further increased median survival in vivo compared to single agents alone. This study thus provides a clinically predictive and personalized drug-testing platform that identifies new treatment opportunities for GBM, warranting further investigation. Citation Format: Sohyon Lee, Tobias Weiss, Marcel Bühler, Rebekka Wegmann, Julien Mena, Michel Bihl, Sandra Goetze, Audrey van Drogen, Elisabeth J. Rushing, Bernd Wollscheid, Michael Weller, Berend Snijder. Image-based functional precision medicine for repurposing neuroactive drugs in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5325.

Abstract 1843: Dianhydrogalactitol (VAL-083) for the treatment of glioblastoma multiforme (GBM): Impact of glucose transporters for crossing the blood brain barrier (BBB)

Abstract Glucose transporters at the blood brain barrier (BBB) maintain the continuous high glucose and energy demands of the brain. These transporters have been targets for the creation of therapeutic molecules as a means to cross the BBB. The BBB has been a major limitation for the successful development of treatments for brain tumors. To date, only a limited number of lipophilic small chemical entities, which can cross the BBB, have been approved for the treatment of GBM. These include the nitrosoureas such as carmustine (BCNU) and lomustine (CCNU) and temozolomide (TMZ). VAL-083 is a di-epoxide substituted hexitol sugar (galactitol). It has a molecular weight of 146 and is highly water soluble. VAL-083 is active against several GBM cell lines and animal models and acts as a DNA targeting agent rapidly forming cross-links at the N7 position of guanine, resulting in lethal DNA double strand breaks and cancer cell death. Its is active in GBM patients, both newly diagnosed and recurrent. Furthermore, its activity is independent of the DNA repair enzyme MGMT which limits the effectiveness of O6 guanine alkylating agents such as the nitrosoureas and TMZ.In clinical pharmacology studies, VAL-083 rapidly accumulates in the cerebrospinal fluid (CSF) and brain tumor tissue. Studies with radiolabeled VAL-083 have shown a half-life of ~ 20 h in CSF vs 2h for plasma post-IV infusion. Recent clinical studies in newly diagnosed GBM patients demonstrated levels of VAL-083 in CSF at least as high as in plasma two hours post infusion (ratio 1.24 ± 0.35). In comparison, peak TMZ concentrations in the CSF are ~30% of that of the plasma.Due to its structural similarity with glucose, we have initiated in vitro glucose transporter studies to identify if SGLTs (sodium dependent glucose cotransporters) or GLUTs (glucose transporters) are contributing to brain penetration. Our initial studies evaluated VAL-083 as a substrate of the human SGLT1 and SGLT2 SLC (solute carrier) transporters. HEK293 cells expressing either the SGLT1 or SGLT2 transporter were exposed to VAL-083 at 3 and 15 µM for 2 and 20 min. The probe substrate methyl-α-D- glucopyranoside (AMG) was used as a positive control, as well as comparison against the reference inhibitors phloridzin dihydrate (100 µM, 20 min) for SGLT1 and dapagliflozin (0.3 µM, 10 min) for SGLT2. Surprisingly, no transporter-specific accumulation of VAL-083 by SGLT1 or SGLT2 was observed. Ongoing studies in CaCo-2 cells are investigating whether VAL-083 is transported via other glucose transporters including GLUT1. GLUT1 is expressed on erythrocytes and the endothelial cells of the BBB and may be highly relevant to the uptake of VAL-083 into brain tissue. The results of these studies will be reported at the meeting. Citation Format: John M. Langlands, Sarath Kanekal, Anne Steino, Armando Mendez, Zoltan Karman, Beata Martonne Toth, Krisztina Heredi-Szabo, Dennis Brown. Dianhydrogalactitol (VAL-083) for the treatment of glioblastoma multiforme (GBM): Impact of glucose transporters for crossing the blood brain barrier (BBB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1843.

Abstract LB136: Characterization of a novel PI3Kinase inhibitor for treatment of glioblastoma multiforme

Abstract The purpose of this study was to test the effects of a novel small molecule inhibitor of phosphatidylinositol-3 kinase (PI3K) for the treatment of glioblastoma multiforme (GBM). The key hurdles to effective treatment for GBM, the “most deadly” form of brain cancer, are resistance to chemotherapy, to radiation, and to immune check point inhibitor therapy. The overarching goal of our experiments has been to determine if a well-characterized small molecule inhibitor of PI3K, GCT.Glio.1, would improve treatment outcomes for GBM patients. The PI3K pathway has been shown to confer both chemotherapy and radiation resistance in GBM. GCT.Glio.1 (NPT520-337), a small molecule compound known to cross the blood brain barrier (BBB), was developed to target PI3K to treat GBM. GBMs, like other “difficult to treat” tumors, often over-express and actively utilize the PI3K pathway for survival, cell growth, and cell division. We used this small molecule inhibitor to determine how targeting the PI3K pathway impacts treatment resistance in GBM. We hypothesized that targeting PI3K would result in growth arrest, increase cell surface expression of a key immune check-point inhibitor target PD-L1, and would sensitize the GBM to treatment with radiation. Treatment of GL261 mouse GBM and U251 human GBM cell lines with GCT.Glio.1 resulted in profound growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1, and synergized with radiation to cause growth arrest and subsequent cell death. In conclusion, these results indicate that GCT.Glio.1 may be a strong candidate for treatment of GBM, and suggest that GCT.Glio.1 is a candidate for rationally designed combinations with currently available radiation and immune checkpoint inhibitor therapies. Currently, the animal studies using GCT.Glio.1 in the GL261 mouse model of GBM have been initiated. Based on our promising pre-clinical data and early encouraging results from pre-clinical pharmacology and safety studies, we anticipate filing a pre-Investigational New Drug application (pre-IND) as the next step toward our goal of a clinical trial using GCT.Glio.1 for GBM patients. Citation Format: Ekokobe Fonkem, M. Karen Newell-Rogers, Richard Tobin, Debbie Healey, Mita Das, Sara Bowen, Chad Quarles, John Clark. Characterization of a novel PI3Kinase inhibitor for treatment of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB136.

Abstract 3895: Moxidectin unravels the role of Hippo-YAP pathway in maintaining immunity of pediatric glioblastoma

Abstract Pediatric glioblastoma multiforme (GBM) is considered to be the second most lethal childhood cancer type after leukemia. Recent preclinical, clinical and genomic studies have highlighted upon the role of Hippo-Yap pathway in the progression of pediatric GBM. In addition, recent studies have established the role of YAP in creating immune suppressive tumor microenvironment (TME) facilitating drug resistance, recurrence and metastasis of GBM tumors. Herein, we report that ‘moxidectin’ an anti-helminthic drug inhibits the proliferation of SF268, SF295, SF188 and CT-2A Luc GBM cells by inducing apoptosis. Immunoblotting and immunofluorescence microscopy studies show that moxidectin mediates its effects by inhibiting MEK-ERK pathway, a regulator of Hippo-YAP signaling. As a result, moxidectin suppressed the nuclear translocation and transcriptional activity of YAP/TAZ-TEAD complex. Oral administration of 3.5mg/kg moxidectin suppressed the growth of GBM tumors by 90% in intracranial tumor model. Ex-vivo analysis of excised tumors confirmed the observations made in in vitro studies. We further conducted immunophenotyping on the excised tumors from both control and moxidectin treated mice to evaluate the effect of moxidectin on immune cell markers in TME and Tumor Draining Lymph Nodes (TDLNs). Our analysis indicated that treatment with moxidectin suppressed the immune suppressive TME created by GBM tumor. Interestingly, moxidectin enhanced antigen presentation in the TDLN. Indicating that it might play a role in activating the peripheral immune response. Moxidectin is an FDA approved drug and any findings from our research will promote its further investigation as a potential therapeutic agent for GBM patients. Citation Format: Itishree Kaushik, Shreyas Gaikwad, Sanjay K. Srivastava. Moxidectin unravels the role of Hippo-YAP pathway in maintaining immunity of pediatric glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3895.

Abstract CT569: Combination of reovirus (pelareorep) and granulocyte-macrophage colony-stimulating factor (GM-CSF) alongside standard chemoradiotherapy and adjuvant chemotherapy (temozolomide) for patients with glioblastoma multiforme (GBM): Long term follow up results of the ReoGlio phase Ib trial

Abstract Background: Oncolytic viruses are of increasing interest as an immunologic approach to treating glioma. We previously reported safety data from a phase Ib, open-label study of intravenous pelareorep with GM-CSF alongside standard chemoradiotherapy in newly diagnosed glioblastoma and confirmed that the combination is tolerable. Following agreement from the relevant ethics authorities we have now completed long term follow up data on all patients treated in the study to investigate whether there is a signal of impact on survival. METHODS: 15 patients with newly diagnosed GBM were treated with GM-CSF 50μg subcutaneously on days 1-3 and pelareorep on days 4-5 in weeks 1 and 4 of chemoradiotherapy, and subsequently in week 1 of each adjuvant temozolomide course: 7 patients received 1x1010TCID50 (dose level 1); 8 received 3x1010TCID50 (dose level 2). The primary objective was to determine the maximum tolerated dose of pelareorep and GM-CSF with standard chemoradiotherapy. Following end of study patients were followed up as per institutional practice. Ethical approval was granted to collect survival data in all patients who survived beyond study closure up to June 2021. Results: We showed that using intravenous pelareorep with GM-CSF alongside standard chemoradiotherapy in patients with GBM was tolerable with 87% of patients completing treatment as planned. Survival data analysis showed that median OS was 12.6 months for patients in dose level 1 and 16.1 months in dose level 2, giving median OS for all patients 13.1 months. It was notable however that a small number of patients survived beyond 24 months. The 24-month survival estimate for all patients was 33%, 16.7% for dose level 1 and 50% for dose level 2. One patient in dose level 2 remains alive at 42 months. Conclusion: We previously reported that intravenous delivery of pelareorep with standard chemoradiotherapy is tolerable in newly diagnosed GBM. Although based on small numbers, these long-term follow up data suggest that this may be an active combination in a subset of GBM patients and further randomized studies are warranted. Citation Format: Susan C. Short, Jessica Kendall, Anthony Chalmers, Catherine McBain, Alan Melcher, Adel Samson, Rachel Phillip, Sarah Brown. Combination of reovirus (pelareorep) and granulocyte-macrophage colony-stimulating factor (GM-CSF) alongside standard chemoradiotherapy and adjuvant chemotherapy (temozolomide) for patients with glioblastoma multiforme (GBM): Long term follow up results of the ReoGlio phase Ib trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT569.

Abstract 5922: Clinical validation of a spectroscopic liquid biopsy for early detection of brain cancer

Abstract Diagnostic delays impact the quality of life and survival of patients with brain tumors. Earlier and expeditious diagnoses in these patients are crucial to reducing the morbidities and mortalities associated with brain tumors. A simple, rapid blood test that can be administered easily in a primary care setting to efficiently identify symptomatic patients who are most likely to have a brain tumor would enable quicker referral to brain imaging for those who need it most. Blood serum samples from 603 patients were prospectively collected and analyzed. Patients either had non-specific symptoms that could be indicative of a brain tumor on presentation to the Emergency Department, or a new brain tumor diagnosis and referral to the neurosurgical unit, NHS Lothian, Scotland. Patient blood serum samples were analyzed using the Dxcover® Brain Cancer liquid biopsy. This technology utilizes infrared spectroscopy combined with a diagnostic algorithm to predict the presence of intracranial disease. Our liquid biopsy approach reported an area under the receiver operating characteristic curve of 0.8. The sensitivity-tuned model achieves a 96% sensitivity with 45% specificity (NPV 99.3%) and identified 100% of glioblastoma multiforme patients. When tuned for a higher specificity, the model yields sensitivity of 47% with 90% specificity (PPV 28.4%). This simple, non-invasive blood test facilitates the triage and radiographic diagnosis of brain tumor patients, while providing reassurance to healthy patients. Minimizing time to diagnosis would facilitate identification of brain tumor patients at an earlier stage, enabling more effective, less morbid surgical and adjuvant care. Citation Format: James M. Cameron, Paul M. Brennan, Georgios Antoniou, Holly J. Butler, Loren Christie, Justin J.A. Conn, Tom Curran, Ewan Gray, Mark G. Hegarty, Michael Jenkinson, Daniel Orringer, David S. Palmer, Alexandra Sala, Benjamin R. Smith, Matthew J. Baker. Clinical validation of a spectroscopic liquid biopsy for early detection of brain cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5922.

Association of plasma levetiracetam concentration, MGMT methylation and sex with survival of chemoradiotherapy-treated glioblastoma patients

Glioblastoma multiforme (GBM) is an aggressive brain tumor, often occurring with seizures managed with antiepileptic drugs, such as levetiracetam (LEV). This study is aimed at associating progression-free survival (PFS) and overall survival (OS) of GBM patients with LEV plasma concentration, MGMT promoter methylation, and sex. In this retrospective, non-interventional, and explorative clinical study, GBM patients underwent surgery and/or radiotherapy and received LEV during adjuvant temozolomide (TMZ) treatment. A high-performance liquid chromatography with UV-detection was used for therapeutic drug monitoring of LEV plasma concentrations. Follow-up average drug concentration was related to patients' clinical characteristics and outcomes. Forty patients (42.5 % female; mean age=54.73 ± 11.70 years) were included, and GBM MGMT methylation status was assessed. All were treated with adjuvant TMZ, and LEV for seizure control. Patients harboring methylated MGMT promoter showed a longer median PFS (460 vs. 275 days, log-rank p < 0.001). The beneficial effect of MGMT promoter methylation was more evident for females (p < 0.001) and in patients with LEV concentration ≤ 20.6 µg/mL (562 days vs. 274.5 days, p = 0.032). Female patients also showed longer OS (1220 vs. 574 days, p = 0.03). Also, higher LEV concentration (>20.6 µg/mL) synergized with MGMT promoter methylation by extending the OS (1014 vs. 406 days of patients with no methylation and low LEV average concentration, p = 0.021). Beneficial effect of higher LEV plasma levels was more evident in males (p = 0.024). Plasma concentrations of LEV may support better outcomes for chemoradiotherapy when other positive prognostic factors are lacking and may promote overall survival by synergizing with MGMT promoter methylation and male sex.

Prognostic utility of lncRNAs (LINC00565 and LINC00641) as molecular markers in glioblastoma multiforme (GBM)

Background and aimGlioblastoma multiforme (GBM) is primary brain tumor grade IV characterized by fast cell proliferation, high mortality and morbidity and most lethal gliomas. Molecular approaches underlying its pathogenesis and progression with diagnostic and prognostic value have been an area of interest. Long-non coding RNAs (lncRNAs) aberrantly expressed in GBM have been recently studied. The aim is to investigate the clinical role of lncRNA565 and lncRNA641 in GBM patients.Patients and methodsBlood samples were withdrawn from 35 newly diagnosed GBM cases with 15 healthy individuals, then lncRNA565 and lncRNA641 expression were evaluated using real time-PCR. Their diagnostic efficacy was detected using receiver operating characteristic curve. Progression free survival (PFS) and overall survival (OS) were studied using Kaplan–Meier curves.ResultslncRNAs expressions were increased significantly among GBM as compared to control group. Their expressions were correlated with clinico-pathological data and survival pattern for the studied GBM patients. Higher levels of both lncRNAs were correlated to worse performance status. Expression of lncRNA565 was increased with large tumor size (≥ 5 cm). Survival analysis showed that both investigated lncRNA were increased with worse PFS and OS.ConclusionExpression of lncRNA565 and lncRNA641 in a liquid biopsy sample can be used as prognostic biomarker for GBM patients.