Primary Glioblastoma Multiforme Cells Research Articles

CHARACTERISATION OF THE RESPONSE OF PRE-CLINICAL MODELS OF GLIOMA TO METABOLIC THERAPIES USING IN VIVO MAGNETIC RESONANCE SPECTROSCOPY

Abstract AIMS Magnetic resonance imaging (MRI) is routinely used to diagnose and monitor brain tumours. Magnetic resonance spectroscopy (MRS) allows us to record the metabolic profile of tumours in situ. This provides a non-invasive method to monitor the response of tumours to metabolic therapies. We aimed to characterise the response of pre-clinical models of glioma to metabolic therapies such as arginine deprivation and the ketogenic diet (KD) by MRS, to better understand what metabolic changes are occurring in the tumour. METHOD We performed orthotopic injection of three different cell lines, including the syngeneic models GL261 and CT2A, as well as a primary GBM cell line, into the striatum of C57BL/6 and athymic nude mice respectively. Mice were randomised to either arginine deprivation treatment using pegylated arginine deiminase (ADI), or KD, plus controls. In each animal, we collected single voxel MRS data using STEAM on both the tumour itself, and healthy brain. MRS data was exported in jMRUI format and analysed in R using the spant package, followed by PCA and OPLS-DA. RESULTS PCA showed that there is clear distinction between healthy and tumour spectra across all models. OPLS-DA indicated that the lipid and choline spectral regions were significantly associated with tumour, whereas the NAA and glutamine/glutamate regions were significantly associated with healthy brain. Integration of specific peaks associated with these metabolites confirmed these results. Subset analysis indicated distinct metabolic signatures between the syngeneic models and the primary GBM model. Furthermore, there is evidence to suggest that tumours in animals treated with arginine deprivation have a unique metabolic signature. CONCLUSION This data indicates that the tumour specific response to metabolic therapies can be measured by routine MRS screening. This would allow a non-invasive method by which clinicians could monitor the effectiveness of metabolic therapies in patients with gliomas.

Abstract LB272: Sequential inhibition of PARP and BET as a rational therapeutic strategy for glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM), characterized as one of the most aggressive brain cancers, presents significant treatment challenges due to its rapid growth, heterogeneity, and robust defensive mechanisms. Despite advancements in neuro-oncology, GBM's prognosis remains grim, with the median survival of approximately 15 months, underscoring the urgent need for innovative therapeutic strategies. Facing these challenges, PARP inhibitors (PARPi) hold substantial promise in treating GBM, evidenced by over 20 ongoing clinical trials. However, their application has been limited by side effects such as nausea, fatigue, and particularly anemia, as well as a narrow therapeutic spectrum focusing mainly on patients with homologous recombination deficiency (HRDness). Through unbiased transcriptomic and proteomic sequencing, and employing Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA), and Transcriptome-Proteome Correlation Analysis, we discovered that the BET inhibitor (BETi) Birabresib profoundly alters the processes of DNA replication and cell cycle progression in GBM cells, extending beyond the previously known impact of BET inhibition on homologous recombination repair. In vitro experiments using GBM cell lines and patient-derived primary GBM cells demonstrated that concurrent administration of PARPi and BETi can synergistically inhibit GBM. Intriguingly, we observed that DNA damage persists after the discontinuation of PARPi monotherapy. Leveraging these residual effects of PARPi, subsequent BETi administration elevated replication stress levels, leading to the inactivation of GBM cell cycle checkpoints, with efficacy comparable to concurrent treatment. The treatment-induced prolonged stalling of replication forks resulted in their collapse and the formation of double-ended double-strand breaks (DSBs). In GBM cells with elevated baseline replication stress, the sequential regimen exhibits comparable efficacy to concurrent treatment, while protecting normal glial cells with lower baseline replication stress from DNA toxicity and subsequent death. In vivo orthotopic transplantation tumor experiments in zebrafish and nude mice demonstrated consistent effects, indicating that sequential administration of PARPi followed by BETi maintained anti-tumor efficacy, similar to concurrent treatment, while reducing toxicity, as evident by the reduced impact on hemoglobin levels. Our findings provide a broader and deeper understanding of the synergistic interaction between BETi and PARPi than previously recognized. This study offers compelling preclinical evidence supporting the development of innovative drug administration strategies focusing on PARPi for GBM therapy. Citation Format: Xin Peng, Xin Huang, Shaolu Zhang, Naixin Zhang, Shengfan Huang, Yingying Wang, Zhenxing Zhong, Shan Zhu, Haiwang Gao, Zixiang Yu, Xiaotong Yan, Zhennan Tao, Yuxiang Dai, Zhe Zhang, Xi Chen, Feng Wang, Francois X. Claret, Ning Ji, Yuxu Zhong, Dexin Kong. Sequential inhibition of PARP and BET as a rational therapeutic strategy for glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB272.

Abstract 5564: The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling

Abstract Glioblastoma (GBM), the most aggressive brain cancer which is usually fatal within a year after diagnosis, poses a significant therapeutic challenge with conventional treatments providing limited success. Despite advancements in immune checkpoint blockade, PD-L1 inhibitors exhibit suboptimal efficacy in recurrent GBMs with recent clinical trials demonstrating only 8% objective responses in patients. Utilizing the R2 Genomic Analysis Visualization Platform, we identified a positive 8-18% correlation between Pyk2 and PD-L1 gene expression in human GBM specimens. We hypothesize that Pyk2 signaling is involved in the regulation of PD-L1 expression in GBM cells. In this study, primary human glioblastoma cell lines and GL261 mouse glioma cells were assessed employing CRISPR/Cas9 Pyk2 knockout (Pyk2KO) combined with pharmacological Pyk2 inhibition. Flow cytometric analysis revealed a 35% decrease in PD-L1 expression in Pyk2KO GL261 cells compared to wild-type cells. SiRNA knockdown against Pyk2 in primary human GBM cells demonstrated a similar response, with a 50% decrease in PD-L1 expression compared to MOCK control cells. Additionally, the application of the Pyk2/FAK inhibitor defactinib at a concentration of 0.2 μM reduced PD-L1 expression by 32% in both GL261 and primary human GBM cells. These findings highlight Pyk2 as a potential therapeutic target for modulating the immunosuppressive microenvironment in GBM and enhancing the effectiveness of immune checkpoint blockade therapies. This study was supported by: NIH Grant 1SC1GM122691 and PRSTRT2022. Citation Format: Tyrel Porter, Lilia Kucheryavykh. The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5564.

Abstract 4621: Dual targeting as an effective therapeutic strategy for malignant brain tumors

Abstract Glioblastoma, a type of brain cancer known for its aggressive nature and resistance to traditional treatments, remains a significant challenge for the medical community. Despite advances in surgical, radiation, and chemotherapy strategies, patients with glioblastoma typically have a poor prognosis, with a median survival time of only 15 months. While temporary symptom relief may be possible for some patients, complete tumor remission is often not achieved. Given the complexity of this disease, effective treatment options for glioblastoma are highly sought after. In this study, we have evaluated the effectiveness of a combinatorial approach using stem cell-derived exosomes enriched with miR-124 cargo paired with viral oncolysis for treating glioblastoma. Human Umbilical Cord Stem cells (hUCs) are genetically modified with specific miR plasmids, and then cultivated in a specialized medium that has been depleted of exosomes. The exosomes (exo) that contain the target miR are subsequently extracted, purified, quantified, and characterized. These exosomes are then utilized for all subsequent investigations. Using a combination of cell viability assays and microscopy, the overall effect of the exo-miR-124 and oncolytic virus on both established and patient-derived primary GBM cells is analyzed. Additionally, a time course western blot analysis is conducted to comprehend the molecular mechanisms underlying the effects of the miR modification and viral oncolysis on cell proliferation and death pathways. Orthotopic animal models of GBM will be utilized to assess the effects of miR modulation and viral oncolysis in vivo. Our research has shown that stem cell-derived exosomes are an effective way to deliver microRNA to specific cells, including tumor cells. This is because they can evade the immune system and target cells directly. Our experiments have shown that combining miR modulation paired with viral oncolysis as a promising approach for treating glioblastoma. Specifically, we have found that miR modulation and viral oncolysis can halt tumor cell proliferation in vitro by targeting multiple pathways involved in GBM growth and progression. Citation Format: Elle Y. Magnan, Karthik Gourishetti, Sydney Thomas, Deepak Bhere. Dual targeting as an effective therapeutic strategy for malignant brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4621.

Abstract A021: Exosome delivered combinatorial treatment for malignant brain tumors

Abstract Glioblastoma (GBM), a World Health Organization (WHO) Grade IV astrocytoma, is a highly aggressive brain tumor with poor prognosis. Current therapeutic approaches, which include surgery, chemotherapy, and radiation therapy, are largely ineffective and often result in tumor recurrence. Several characteristics of the tumors make them challenging to treat, including their protection by the blood-brain barrier, which hinders effective treatment delivery. Our research addresses this issue by using exosomes, which are small cell-derived vesicles, as delivery vehicles for microRNA (miR) therapies. Dysregulation of miR expression has been implicated in tumorigenesis, and targeting these dysregulated miRs is emerging as a promising approach to tumor treatment. In particular, miR-7 and miR-124 have been identified as tumor-suppressive miRs that are downregulated in various tumors, including GBM. In this study, stem cell-derived exosomes loaded with miR-7 and miR-124 have been evaluated as a combinatorial approach for treating GBM. In our work, human adipose stem cells (ADSCs) were transduced with the target miR-7 and miR-124 plasmids and cultured in exosome-depleted FBS. The miR-containing exosomes from the ADSCs were then isolated, purified, quantified, and characterized for use in subsequent studies. Murine and human GBM cells as well as primary patient-derived primary GBM cells were treated with miR-7 and miR-124 exosomes, and the efficacy of exosome treatment was determined using cell viability assays and microscopy. The mechanisms of miR modulation of cell proliferation and cell death pathways were analyzed with time-course western blot analysis. Orthotopic animal models of GBM will be used to assess the effectiveness of our methods in vivo. Our results have demonstrated successful delivery of miR-7 and miR-124 to GBM cells via stem cell-derived exosomes. Data show that treatment with these tumor-suppressive miRs halts GBM growth in vitro by modulating multiple pathways involved in tumor growth and progression. While we await results of our in vivo studies, our preliminary data demonstrate that delivery of miR-7 and miR-124 via stem cell-derived exosomes is a promising therapeutic approach for treating GBM. Citation Format: Sydney Thomas, Karthik Gourishetti, Karthik Rangavajhula, Deepak Bhere. Exosome delivered combinatorial treatment for malignant brain tumors [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A021.

Transduction Efficiency of Zika Virus E Protein Pseudotyped HIV-1gfp and Its Oncolytic Activity Tested in Primary Glioblastoma Cell Cultures.

The development of new tools against glioblastoma multiforme (GBM), the most aggressive and common cancer originating in the brain, remains of utmost importance. Lentiviral vectors (LVs) are among the tools of future concepts, and pseudotyping offers the possibility of tailoring LVs to efficiently transduce and inactivate GBM tumor cells. Zika virus (ZIKV) has a specificity for GBM cells, leaving healthy brain cells unharmed, which makes it a prime candidate for the development of LVs with a ZIKV coat. Here, primary GBM cell cultures were transduced with different LVs encased with ZIKV envelope variants. LVs were generated by using the pNLgfpAM plasmid, which produces the lentiviral, HIV-1-based, core particle with GFP (green fluorescent protein) as a reporter (HIVgfp). Using five different GBM primary cell cultures and three laboratory-adapted GBM cell lines, we showed that ZIKV/HIVgfp achieved a 4-6 times higher transduction efficiency compared to the commonly used VSV/HIVgfp. Transduced GBM cell cultures were monitored over a period of 9 days to identify GFP+ cells to study the oncolytic effect due to ZIKV/HIVgfp entry. Tests of GBM tumor specificity by transduction of GBM tumor and normal brain cells showed a high specificity for GBM cells.

Expression of molecular markers and synergistic anticancer effects of chemotherapy with antimicrobial peptides on glioblastoma cells.

Glioblastoma multiforme (GBM) is the most aggressive and fatal malignant primary brain tumor. The enhancement of the survival rate for glioma patients remains limited, even with the utilization of a combined treatment approach involving surgery, radiotherapy, and chemotherapy. This study was designed to assess the expression of IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX in glioblastoma tissue from 11 patients. We investigated the anticancer impact and combined effects of cathelicidin (LL-37), protegrin-1 (PG-1), with chemotherapy-temozolomide (TMZ), doxorubicin (DOX), carboplatin (CB), cisplatin (CPL), and etoposide (ETO) in primary GBM cells. In addition, we examined the effect of LL-37, PG-1 on normal human fibroblasts and in the C6/Wistar rat intracerebral glioma model. For this study, 11 cases of glioblastoma were evaluated immunohistochemically for IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cells viability and to determine cytotoxic effects of LL-37, PG-1 and their combination with chemotherapy in primary GBM cells. Synergism or antagonism was determined using combination index (CI) method. Finally, we established C6 glioblastoma model in Wistar rats to investigate the antitumor activity. Peptides showed a strong cytotoxic effect on primary GBM cells in the MTT test (IC50 2-16 and 1-32μM) compared to chemotherapy. The dual-drug combinations of LL-37 + DOX, LL-37 + CB (CI 0.46-0.75) and PG-1 + DOX, PG-1 + CB, PG-1 + TMZ (CI 0.11-0.77), demonstrated a synergism in primary GBM cells. In rat C6 intracerebral GBM model, survival of rats in experimental group (66.75 ± 12.6days) was prolonged compared with that in control cohort (26.2 ± 2.66days, p = 0.0008). After LL-37 treatment, experimental group rats showed significantly lower tumor volumes (31.00 ± 8.8 mm3) and weight (49.4 ± 13.3mg) compared with control group rats (153.8 ± 43.53mg, p = 0.038; 82.50 ± 7.60 mm3, respectively). The combination of antimicrobial peptides and chemical drugs enhances the cytotoxicity of chemotherapy and exerts synergistic antitumor effects in primary GBM cells. Moreover, in vivo study provided the first evidence that LL-37 could effectively inhibit brain tumor growth in rat C6 intracerebral GBM model. These results suggested a significant strategy for proposing a promising therapy for the treatment of GBM.

Dual blockade of EGFR and PI3K signaling pathways offers a therapeutic strategy for glioblastoma

BackgroundGlioblastoma multiforme (GBM) is a devastating disease that lacks effective drugs for targeted therapy. Previously, we found that the third-generation epidermal growth factor receptor (EGFR) inhibitor AZD-9291 persistently blocked the activation of the ERK pathway but had no inhibitory effect on the phosphoinositide 3-kinase (PI3K)/Akt pathway. Given that the PI3K inhibitor GDC-0084 is being evaluated in phase I/II clinical trials of GBM treatment, we hypothesized that combined inhibition of the EGFR/ERK and PI3K/Akt pathways may have a synergistic effect in the treatment of GBM.MethodsThe synergistic effects of cotreatment with AZD-9291 and GDC-0084 were validated using cell viability assays in GBM and primary GBM cell lines. Moreover, the underlying inhibitory mechanisms were assessed through colony formation, EdU proliferation, and cell cycle assays, as well as RNA-seq analyses and western blot. The therapeutic effects of the drug combination on tumor growth and survival were investigated in mice bearing tumors using subcutaneously or intracranially injected LN229 xenografts.ResultsCombined treatment with AZD-9291 and GDC-0084 synergistically inhibited the proliferation and clonogenic survival, as well as induced cell cycle arrest of GBM cells and primary GBM cells, compared to monotherapy. Moreover, AZD-9291 plus GDC-0084 combination therapy significantly inhibited the growth of subcutaneous tumors and orthotopic brain tumor xenografts, thus prolonging the survival of tumor-bearing mice. More importantly, the combination of AZD-9291 and GDC-0084 simultaneously blocked the activation of the EGFR/MEK/ERK and PI3K/AKT/mTOR signaling pathways, thereby exerting significant antitumor activity.ConclusionOur findings demonstrate that the combined blockade of the EGFR/MEK/ERK and PI3K/AKT/mTOR pathways is more effective against GBM than inhibition of each pathway alone, both in vitro and in vivo. Our results suggest that AZD-9291 combined with GDC-0084 may be considered as a potential treatment strategy in future clinical trials.396yfhm1sdcH9_KhVkr-McVideo

TMIC-41. LATERAL VENTRICLE PROXIMITY OF GLIOBLASTOMA IS ASSOCIATED WITH LOCALIZED MALIGNANCY-PROMOTING MOLECULAR SIGNATURES IN IMAGE-GUIDED BIOPSIES

Abstract Glioblastoma (GBM) is the most devastating and common form of primary brain cancer in adults. Tumor location plays a significant role in patient prognosis; in particular, GBM tumors contacting the lateral ventricles (LVs) are more aggressive than LV-distal counterparts. This may be due to interaction of tumors with unique features of the LV microenvironment, including the cerebrospinal fluid (CSF) and neural progenitor cells (NPCs) of the subventricular zone. Despite LV contact having a role on tumor malignancy, studies using bulk tumor samples to identify molecular contributors to LV-contacting prognosis have been only moderately successful, in large part due to intratumoral heterogeneity. Here, we leverage intraoperative surgical navigation to collect matched biopsies of adult LV-contacting primary GBM at locations proximal (< 2 cm) and distal ( > 2 cm) from the LV. Matched samples were collected from ten patients with equal sex distribution and an age range of 46 to 81 years. RNA sequencing of biopsies revealed a localized transcriptional signature of gene expression in LV-proximal locations. These genes include those identified to be associated with GBM malignancy and upregulated in primary GBM cells by CSF exposure or co-culture with NPCs, such as SERPINA3, CD44, and CTSB, as well as many newly identified genes. Implicated biological pathways involved in this transcriptional signature involve inflammation, chemotaxis, Notch signaling, angiogenesis, integrin signaling, and more. The top 50 upregulated genes by LV-proximity of GBM were used to cluster patient transcriptomes from the TCGA GBM database and evaluate the contribution to patient outcome. This analysis revealed a cluster with significantly extended overall survival that displayed a general downregulation of LV-proximity genes. Ultimately, these studies provide evidence that regional tumoral analysis is essential in identifying molecular signatures associated with microenvironmental influence, and that LV-contacting GBM has regional gene expression changes in many genes associated with malignancy.

DDDR-06. THE SMOKING CESSATION AGENT LOBELINE DELAYS HYPOXIC STRESS RECOVERY RESULTING IN GLIOBLASTOMA CELL DEATH

Abstract Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumour with a universally fatal prognosis. GBM is characterized by extensive regions of hypoxia which adversely impacts clinical outcomes, as hypoxia promotes chemoresistance. Tumour cells can adapt to and survive hypoxic stress by inducing rapid changes in the translational landscape, facilitated in part by the formation of stress granules (SGs). SGs are cytoplasmic aggregates of untranslated mRNAs and RNA binding proteins formed in response to a variety of cellular stressors, that allow cells to temporarily prioritize translation of stress-related proteins. Previous work in our laboratory has shown that GBM cells form SGs in vitro and in vivo, and that pharmacologically altering SG dynamics to prevent their dissolution (effectively locking cells in a stressed state) increases GBM cell death. Here we have identified another compound that delays the dissolution of hypoxia-induced SGs in GBM cells, the smoking cessation agent lobeline. SG dissolution typically occurs within 15 minutes post-hypoxia, however pre-treatment of immortalized GBM cells with lobeline delays SG dissolution for up to 2 hours. This delay in SG dissolution is dose dependent, as higher lobeline concentrations resulted in corresponding increases in the percentage of cells containing SGs and the average number of SGs per cell post-hypoxia. Lobeline also led to sustained phosphorylation of eIF2a (a key regulator of translation initiation) with an accompanying decrease in global protein translation levels post-hypoxia. Importantly, the combination of lobeline and hypoxia led to synergistic cell death in both immortalized and primary GBM cells, specifically through increased necrosis. Lobeline acts as a VMAT2 ligand and can stimulate dopamine release. Interestingly, we have found miRNA related to dopamine metabolism pathways overrepresented in extracellular vesicles isolated from GBM patient plasma. We postulate that disrupting the balance of stress pathways may have therapeutic potential in the future.

BIOM-35. IDENTIFICATION AND VALIDATION OF POTENTIAL BIOMARKERS FOR DECITABINE-MEDIATED SENSITIZATION TO TEMOZOLOMIDE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with limited treatment options and a poor prognosis. Our previous research demonstrated that the DNA hypomethylating agent decitabine (DAC) enhances the sensitivity to temozolomide (TMZ) of a subset of primary GBM cell lines derived from fresh surgical tissue specimens, and that this effect may be mediated by upregulation of DNA mismatch repair genes including MLH1. In this context, we aim to identify additional biomarkers for DAC-mediated sensitization to TMZ with potential clinical utility through in vitro and in vivo experiments. We performed single cell RNA sequencing on three DAC-sensitized and three DAC-desensitized cell lines and found 4 genes that exhibited significant differential expression: DCN, LUM, CRABP2 and NUPR1. We validated these findings using quantitative immunoblots, RT-qPCR and immunohistochemistry, finding that NUPR1 protein expression levels varied most significantly between DAC-sensitized and desensitized GBM tissues. Long-range direct methylation sequencing of non-amplified genomic DNA enriched using CRISPR/Cas9 demonstrated several CpG sites within the 4 genes that may be predictive of TMZ sensitization response after treatment with DAC. Finally, we performed mouse survival experiments using orthotopic patient-derived xenografts (PDXs) of the six GBM cell lines, with preliminary results showing that in selected PDX survival was significantly increased with combined DAC-TMZ versus TMZ monotherapy as predicted by in vitro assays. In conclusion, our study demonstrates that combining DAC with TMZ may be a potential treatment strategy for GBM in a subset of patients. There likely exist several biomarkers that can be used at the time of diagnosis, alone or in combination, to predict the response to epigenetic preconditioning using DAC for TMZ sensitization, which will be necessary for optimal patient selection in an early phase clinical trial. Further data and validation studies are necessary to strengthen these findings and to confirm their clinical relevance.

Establishment of a 3D Model to Characterize the Radioresponse of Patient-Derived Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite modern, multimodal therapeutic options of surgery, chemotherapy, tumor-treating fields (TTF), and radiotherapy, the 5-year survival is below 10%. In order to develop new therapies, better preclinical models are needed that mimic the complexity of a tumor. In this work, we established a novel three-dimensional (3D) model for patient-derived GBM cell lines. To analyze the volume and growth pattern of primary GBM cells in 3D culture, a CoSeedisTM culture system was used, and radiation sensitivity in comparison to conventional 2D colony formation assay (CFA) was analyzed. Both culture systems revealed a dose-dependent reduction in survival, but the high variance in colony size and shape prevented reliable evaluation of the 2D cultures. In contrast, the size of 3D spheroids could be measured accurately. Immunostaining of spheroids grown in the 3D culture system showed an increase in the DNA double-strand-break marker γH2AX one hour after irradiation. After 24 h, a decrease in DNA damage was observed, indicating active repair mechanisms. In summary, this new translational 3D model may better reflect the tumor complexity and be useful for analyzing the growth, radiosensitivity, and DNA repair of patient-derived GBM cells.

Changes in calpain-2 expression during glioblastoma progression predisposes tumor cells to temozolomide resistance by minimizing DNA damage and p53-dependent apoptosis

BackgroundGlioblastoma multiforme (GBM) is characterized by an unfavorable prognosis for patients affected. During standard-of-care chemotherapy using temozolomide (TMZ), tumors acquire resistance thereby causing tumor recurrence. Thus, deciphering essential molecular pathways causing TMZ resistance are of high therapeutic relevance.MethodsMass spectrometry based proteomics were used to study the GBM proteome. Immunohistochemistry staining of human GBM tissue for either calpain-1 or -2 was performed to locate expression of proteases. In vitro cell based assays were used to measure cell viability and survival of primary patient-derived GBM cells and established GBM cell lines after TMZ ± calpain inhibitor administration. shRNA expression knockdowns of either calpain-1 or calpain-2 were generated to study TMZ sensitivity of the specific subunits. The Comet assay and ɣH2AX signal measurements were performed in order to assess the DNA damage amount and recognition. Finally, quantitative real-time PCR of target proteins was applied to differentiate between transcriptional and post-translational regulation.ResultsCalcium-dependent calpain proteases, in particular calpain-2, are more abundant in glioblastoma compared to normal brain and increased in patient-matched initial and recurrent glioblastomas. On the cellular level, pharmacological calpain inhibition increased the sensitivities of primary glioblastoma cells towards TMZ. A genetic knockdown of calpain-2 in U251 cells led to increased caspase-3 cleavage and sensitivity to neocarzinostatin, which rapidly induces DNA strand breakage. We hypothesize that calpain-2 causes desensitization of tumor cells against TMZ by preventing strong DNA damage and subsequent apoptosis via post-translational TP53 inhibition. Indeed, proteomic comparison of U251 control vs. U251 calpain-2 knockdown cells highlights perturbed levels of numerous proteins involved in DNA damage response and downstream pathways affecting TP53 and NF-κB signaling. TP53 showed increased protein abundance, but no transcriptional regulation.ConclusionTMZ-induced cell death in the presence of calpain-2 expression appears to favor DNA repair and promote cell survival. We conclude from our experiments that calpain-2 expression represents a proteomic mode that is associated with higher resistance via “priming” GBM cells to TMZ chemotherapy. Thus, calpain-2 could serve as a prognostic factor for GBM outcome.

The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor with a poor median survival of less than 15 months. However, clinical strategies and effective therapies are limited. Here, we found that the second-generation small molecule multi-CDK inhibitor AT7519 is a potential drug for GBM treatment according to high-throughput screening via the Approved Drug Library and Clinical Compound Library (2718 compounds). We found that AT7519 significantly inhibited the cell viability and proliferation of U87MG, U251, and patient-derived primary GBM cells in a dose-dependent manner. Furthermore, AT7519 also inhibited the phosphorylation of CDK1/2 and arrested the cell cycle at the G1-S and G2-M phases. More importantly, AT7519 induced intrinsic apoptosis and pyroptosis via caspase-3-mediated cleavage of gasdermin E (GSDME). In the glioblastoma intracranial and subcutaneous xenograft assays, tumor volume was significantly reduced after treatment with AT7519. In summary, AT7519 induces cell death through multiple pathways and inhibits glioblastoma growth, indicating that AT7519 is a potential chemical available for GBM treatment.

2-Deoxyglucose, an Inhibitor of Glycolysis, Enhances the Oncolytic Effect of Coxsackievirus.

Glioblastoma multiforme (GBM) is one of the most common types of brain tumor. Despite intensive research, patients with GBM have a poor prognosis due to a very high rate of relapse and significant side effects of the treatment, with a median survival of 14.6 months. Oncolytic viruses are considered a promising strategy to eliminate GBM and other types of cancer, and several viruses have already been introduced into clinical practice. However, identification of the factors that underly the sensitivity of tumor species to oncolytic viruses or that modulate their clinical efficacy remains an important target. Here, we show that Coxsackievirus B5 (CVB5) demonstrates high oncolytic potential towards GBM primary cell species and cell lines. Moreover, 2-deoxyglucose (2DG), an inhibitor of glycolysis, potentiates the cytopathic effects of CVB5 in most of the cancer cell lines tested. The cells in which the inhibition of glycolysis enhanced oncolysis are characterized by high mitochondrial respiratory activity and glycolytic capacity, as determined by Seahorse analysis. Thus, 2-deoxyglucose and other analogs should be considered as adjuvants for oncolytic therapy of glioblastoma multiforme.

EXTH-77. TARGETING CD73 IN GBM SENSITIZES TUMORS TO ONCOLYTIC VIRUS THERAPY

Abstract Glioblastoma Multiforme (GBM) is the most aggressive malignant primary brain tumor and has abysmal 5-year overall survival. With the approval of oHSV Imlygic by FDA for metastatic melanoma and more recently, conditional approval of G47Δ, marketed by Daiichi Sankyo for GBM treatment in Japan, oncolytic viral therapy has emerged as a promising biological approach to treat solid tumors. Our laboratory has previously shown that oHSV expressing long isoform of PTEN (PTENα), HSV-P10, has faster kinetics of virus replication associated with enhanced killing of the glioma cells compared to control HSV. Additionally, HSVP-10 is known to increase mitochondrial membrane potential and cellular ATP production while stimulating anti-tumor immune responses in vivo. RNA sequencing of primary GBM cells infected with HSV-P10 shows altered metabolic pathways relative to control HSV infected cells. 13C metabolic flux analysis in uninfected and control or HSV-P10 infected primary GBM cells reveals that while HSV-P10 infection shuttles most of the glutamine towards citrate by reductive carboxylation of α-ketoglutarate resulting in faster replication of HSV-P10, simultaneously it increased glucose utilization and shuttling towards TCA cycle. This corroborates with enhanced mitochondrial activity leading to increased oxidative phosphorylation and increased cellular and extracellular ATP (eATP). eATP binds to purinergic receptors on tumor and immune cells and boosts anti-tumor immunity. However, the use of extracellular ATP in cancer therapy is limited owing to its short half-life and rapid hydrolysis by ectoenzymes CD39 and CD73 into immune-suppressing adenosine. CD73 carries out the conversion of AMP to adenosine, making it a key regulator of this pathway. Using mice with CD73 knocked out globally (CD73KO), we show that combination of CD73 inhibition with HSV-P10 imparts significant survival benefit compared to WT mice treated with HSV-P10. Our findings will further the understanding of oHSV therapy and the role of the ATP/adenosine in the tumor microenvironment.

EXTH-51. HSV-PTENΑ TREATMENT TARGETS GLIOMA STEM CELLS TO REDUCE STEMNESS AND SENSITIZES GLIOBLASTOMA TO IRRADIATION

Abstract Glioblastoma is one of the most lethal and treatment-resistant tumors of the central nervous system. Despite maximal surgical and medical therapy, survival remains dismal with a median of 21 months. Despite advances in treatment, this has only led to modest survival benefit. A significant challenge in treatment of glioblastoma is targeting glioma stem cells (GSCs) which are a source of therapy resistance. Oncolytic viral (OV) therapy is a promising therapy for solid tumors that preferentially targets tumor cells for lysis and an anti-tumor immune response while sparing normal cells. Among all OVs, oncolytic Herpes Simplex Virus (oHSV) is substantially ahead in the clinic, with an oHSV T-VEC approved by the FDA for metastatic melanoma treatment and G47∆ which received conditional approval for the treatment of GBM in Japan. In prior work, our group has demonstrated that PTENα expression by an oHSV (HSV-P10) results in improved long-term survivors in intracranial tumor-bearing mice compared to HSVQ treatment. We aim to elucidate the mechanism of improved therapeutic efficacy of HSV-P10 against GBM and evaluate if HSV-P10 may overcome radio-resistance. RNA sequencing and GSEA analysis of primary human GBM cells infected with control HSVQ or HSV-P10 reveals that while HSVQ virus infection leads to an increase in genes regulating IL6-STAT3 pathway, pivotal in maintaining stemness properties, HSV-P10 infection leads to a reduction. HSV-P10 reduces CD133+/CD44+ stem cells, induces DNA damage and sensitizes the GBM cells to irradiation. Our findings reveal a novel mechanism induced by HSV-P10 in combination with irradiation where HSV-P10 modulates IL6-STAT3 signaling downregulating genes associated with stemness (Nestin, Sox2). HSV-P10 infection in combination with irradiation reduces GSC tumor sphere formation in vitro and sensitizes GBMs to radiotherapy in an intracranial mouse xenograft model. Our findings uncover a possible mechanism to overcome GSC-mediated therapy resistance to improve the therapeutic efficacy for GBM.

Transcriptome Analysis of Human Glioblastoma Cells Susceptible to Infection with the Leningrad-16 Vaccine Strain of Measles Virus.

Glioblastoma multiforme (GBM) accounts for almost half of all primary malignant brain tumors in adults and has a poor prognosis. Here we demonstrated the oncolytic potential of the L-16 vaccine strain of measles virus (MV) against primary human GBM cells and characterized the genetic patterns that determine the sensitivity of primary human GBM cells to oncolytic therapy. MV replicated in all GBM cells, and seven out of eight cell lines underwent complete or partial oncolysis. RNA-Seq analysis identified about 1200 differentially expressed genes (FDR < 0.05) with at least two-fold expression level change between MV-infected and uninfected cells. Among them, the most significant upregulation was observed for interferon response, apoptosis and cytokine signaling. One out of eight GBM cell lines was defective in type I interferon production and, thus, in the post-interferon response, other cells lacked expression of different cellular defense factors. Thus, none of the cell lines displayed induction of the total gene set necessary for effective inhibition of MV replication. In the resistant cells, we detected aberrant expression of metalloproteinase genes, particularly MMP3. Thus, such genes could be considered intriguing candidates for further study of factors responsible for cell sensitivity and resistance to L-16 MV infection.

TRIBBLES1 (TRIB1) Pseudokinase Induces Treatment Resistance in GBM by Promoting Survival of Tumor Cells through the Akt Pathway

<h3>Purpose/Objective(s)</h3> Glioblastoma (GBM; WHO grade IV) is the most aggressive form of glioma with a dismal 5-year survival rate. The current therapies are largely ineffective and therapy resistance is commonly encountered. Therefore, there is an urgent need to identify druggable therapeutic targets in GBM. In this study we identified TRIB1, a Ser/Thr pseudokinase that functions as a scaffold to initiate Ubiquitin Proteasome System-mediated degradation of target proteins in the cell. It has also been shown to activate the MAPK and Akt signaling cascades in various cell types. Reports also suggest that TRIB1 contributes to chemotherapy resistance in various cancers. Therefore, we evaluated the role of TRIB1 in GBM cell proliferation/ survival and its contribution to therapy resistance. <h3>Materials/Methods</h3> We utilized patient-centered reverse translational approach to identify novel therapeutic targets. To this end, TRIB1 was identified by statistical association (Cox regression analysis) of the patient-derived mRNA profiling data publicly available from TCGA GBM cohort. TRIB1 was functionally validated <i>in vitro</i> by generating stable overexpression cell lines by antibiotic selection. TRIB1 was conditionally knocked down by doxycycline induction. Co-immunoprecipitation was performed to evaluate protein-protein interactions. Protein levels were detected by western blotting. Site-directed mutagenesis was performed to mutate specific amino acid. Patient derived primary cell lines overexpressing the wild type and mutant (W337A) Trib1 transgenes were injected intracranially to create tumors in nude mice. Changes in tumor volume and overall survival (OS) were determined. <h3>Results</h3> The mRNA profiling of TCGA GBM cohort revealed that increased <i>TRIB1</i> gene expression was associated with worse OS of GBM patients (HR=1.3 (1.0-1.5); P=0.019). Mice bearing TRIB1 transgene overexpressing tumors had highest tumor volume and lowest OS whereas mice bearing TRIB1-W337A tumors had the highest OS and lower tumor volume when measured. We also observed that overexpression of TRIB1 caused a decrease in apoptosis of primary GBM cell lines after RT and TMZ treatments <i>in vitro</i>. Overexpression of TRIB1 also increased the phosphorylation/activation of Akt in patient derived primary cell lines. Akt activation was similarly decreased after TRIB1 knockdown. TRIB1 bound directly to Akt in these cells. Following PI3K inhibition, Akt phosphorylation was detected to a greater level in TRIB1 overexpressing cells. <h3>Conclusion</h3> Our data suggest that heightened expression of TRIB1 in GBM cells contributes to RT/TMZ resistance by activating the prosurvival Akt signaling cascades. Therefore, targeting TRIB1 mediated Akt activation could provide a therapeutic benefit over targeting Akt directly, which may reduce Akt's oncogenic signal without risking the toxicity associated with available Akt- specific inhibitors.

Abstract 5498: Plinabulin, a novel tubulin targeting agent, collapses the proinvasion tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival

Abstract Glioblastoma multiforme (GBM) is the most common adult primary brain malignancy. Aggressive, diffuse invasion is regulated by a dynamic cytoskeleton and significantly contributes to poor survival. Intraparenchymal tumor cell dissemination prevents complete surgical resection and compromises essential brain functions. Invasive cells are inherently less sensitive to traditional cytotoxic therapies. Recent investigation into targeted therapies has failed to address invasion/cytoskeleton based GBM therapy resistance. Furthermore, established GBM cell line modeling in 2 or 3 dimensions (3D) does not capture the genetic diversity or clinical relevance of patient derived primary (1') GBM cell lines, which may uncover/predict differential responses in tumors to novel therapies. Cytoskeleton remodeling and cell invasion in patient-derived primary GBM cells generates unique structures called tumor microtubes (TMs) not seen in established, cultured GBM cell lines. TMs are ultralong, pro-invasive, tubulin-enriched protrusions that promote chemo- and radioresistance in GBM patient derived primary cells. Using our extensive GBM cell line biobank derived from 1' and/or therapy resistant recurrent (2') tumors, we investigated the effects of a novel drug targeting the microtubule cytoskeleton. Plinabulin is a tubulin targeting agent that crosses the blood brain barrier and demonstrated anticancer efficacy in Phase III trial patients with non-small cell lung carcinoma. Plinabulin also has antiproliferative properties in 2D GBM cells. Here, we evaluate the efficacy of plinabulin treatment upon 3D neurosphere viability and invasion in 5 GBM 1' and 2' recurrent patient derived cell lines. Within 24h of matrigel embedding, 3D GBM patient neurospheres generate robust pro-invasive TMs. Plinabulin treatment (10-30 nM) at embedding followed by drug washout after 24h eliminated existing TMs and inhibited single cell invasion (&amp;gt;80% relative to controls) in both 1' and 2' GBM patient neurospheres. Cell survival was significantly reduced in plinabulin treated neurospheres (&amp;gt;30 nM). Plinabulin efficacy was evaluated in a clinically relevant therapeutic ITR model: GBM patient neurospheres invaded (I) for 48h to establish TM networks; were treated (T) with 1-50 nM plinabulin for 24h prior to drug washout; and recovered (R) for 96h. In 1', 2', or matched 1' and 2' cell line pairs from the same patient, 10-40 nM plinabulin sustained invasion inhibition (&amp;gt;90%) through 96h post washout and completely blocked TM extension relative to controls, consistent with established clinical concentrations of plinabulin. This study reveals plinabulin as a potent GBM therapeutic targeting pro-invasion TM networks and survival in 1' and therapy resistant 2' GBM patient cell lines. Citation Format: Kathryn M. Eisenmann, Kenneth Lloyd, Krista M. Pettee, Kathryn N. Becker, Jason Schroeder, Kevin Reinard, James R. Tonra, Ramon Mohanlal, Lan Huang. Plinabulin, a novel tubulin targeting agent, collapses the proinvasion tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival [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 5498.