Primary Glioblastoma Cell Lines Research Articles

Hypofractionated Radiation Therapy Suppresses Radioresistance in U87 Human Glioma Cells by Inhibiting Yap1 and Hsp90 Proteins.

Radiotherapy plays a vital role in the management of high-grade gliomas. However, the radio resistance of glioma cells limits the effect of radiation and drives recurrence inside the irradiated tumor volume leading to poor outcomes for patients. High-grade glioma cell radioresistance significantly contributes to radiotherapy failure, highlighting the importance of identifying predictive biomarkers for radioresistance. An increasing body of evidence complies with the Yes Associated Protein 1 (Yap-1) and heat shock protein 90 (Hsp90) as biomarkers for radioresistance in glioma cells. A number of studies suggest the potential of radioresistance-associated factors as biomarkers and/ or novel therapeutic targets in glioma cells. Thus, it is essential for glioblastoma patients to identify robust druggable targets involved in radioresistance, optimizing irradiation protocol, and understanding their underlying molecular mechanisms. Therefore, in the present study, we hypothesized that hypofractionated Gamma Knife radiation therapy (HF-GKRT) could target Yap-1 and Hsp90 and downregulate the mechanism of radioresistance in high-grade glioma cells. For this purpose, expression levels of radioresistance markers Yap-1 and Hsp90 were evaluated after treatment with HF-GKRT, and this was compared with single fraction Gamma Knife radiation therapy (SF-GKRT) in U87MG primary human glioblastoma cell line model. This would help design a novel radiation therapy regimen for glioblastoma patients by reducing the risk of radioresistance.

OMA1 competitively binds to HSPA9 to promote mitophagy and activate the cGAS–STING pathway to mediate GBM immune escape

BackgroundImmunotherapy with checkpoint inhibitors, especially those targeting programmed death receptor 1 (PD-1)/PD-1 ligand (PD-L1), is increasingly recognized as a highly promising therapeutic modality for malignancies. Nevertheless, the efficiency of immune...

Abstract 55: CD70-specific CAR-T cell therapy for the treatment of glioblastoma

Abstract Despite the remarkable success of chimeric antigen receptor (CAR)-T cell treatment for patients with hematologic malignancies, this method has yet failed to confer meaningful survival benefits to patients suffering from glioblastoma (GB), the most lethal type of brain tumor. This highlights the need to develop novel CAR-T cell approaches for this disease. CD70 is a member of the tumor necrosis factor receptor (TNFR) superfamily. Although absent on normal brain tissue, it is ectopically expressed in a substantial fraction of GB patients, indicating its suitability as a CAR-T cell therapy candidate. In this study, we generated CD70-targeting CAR-T cells and tested their cytotoxicity in vitro and in vivo. First, we detected CD70 in a panel of primary GB cell lines and to investigate its role, it was overexpressed in human and murine GB cells. In a syngeneic glioma model, C57BL/6J mice injected with CD70-overexpressing GL261 cells developed significantly larger tumors compared to the control counterparts. Additionally, RNA-sequencing revealed that CD70-overexpression in the same cells led to higher expression levels of immunosuppressive marker CD200 and lower levels of tumor inhibition genes Prkg2 and Sh3bgrl2, suggesting a tumor-promoting role in GB. For our immunotherapeutic intervention, we designed CD70-targeting CAR-T cell constructs featuring different co-stimulatory domains (CD27, CD28 or 4-1BB) and successfully transduced primary T-Cells from healthy donors. In an in vitro co-culture, all CAR-T cells recognized and eliminated primary cancer cells in a target-dependent and donor-independent manner, while secreting high levels of Granzyme B. The killing capacity of these cells was further highlighted in a 3D system in which in vitro-generated cortical organoids were treated with CAR-T cells after being infiltrated by CD70-expressing or control cells. Immunofluorescence (IF) staining and enzyme-linked immunosorbent assay (ELISA) revealed increased levels of Granzyme B and IFN-γ in all treated organoids previously infiltrated by CD70+ tumor cells. Importantly, generated CAR-T cells showed high specificity and efficiency in killing CD70+ tumors in vivo in the brains of immunodeficient mice. Namely, 80% of NSG mice orthotopically implanted with CD70+ GB cells and subsequently treated by CARs but not mock-transduced T-Cells showed complete tumor remission by the end of the experimental endpoint, determined by bioluminescence imaging (BLI). IF analysis of these brains showed high levels of apoptotic marker cleaved caspase-3, enhanced effector cell presence and significantly lower tumor cell occupancy compared to control-treated animals. In conclusion, we provide evidence in favor of utilization of CAR-T cells against CD70-expressing gliomas. Based on these findings, a phase-I clinical trial to assess the safety and efficacy of autologous CD70-specific CAR-T cells for relapsed CD70+ GB is being planned. Citation Format: Alexandros Kourtesakis, Hiu Nam Hannah Chow, Dennis Alexander Agardy, Eileen Bailey, Sandra Horschitz, Ammar Jabali, Rainer Will, Denise Reibold, Sonja Pusch, Christoph Schifflers, Manuel Fischer, Ling Hai, Dirk C. Hoffmann, Yu-Chan Chih, Robin Wagener, Leon Kaulen, Philipp Koch, Michael Breckwoldt, Michael Schmitt, Wolfgang Wick, Tim Sauer, Tobias Kessler. CD70-specific CAR-T cell therapy for the treatment of glioblastoma [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 55.

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 4422: Metabolic adaptations of glioblastoma in hypoxia is mediated by acox-1

Abstract Background: Glioblastoma (GBM) is a recurrent, chemoresistant, malignant brain tumor. Hypoxia is a major factor contributing to resistance in GBM which develops due to aggressive tumor growth and chronic anti-angiogenic therapy. Thus, under limited oxygen supply it is unlikely that the high energy consuming pathways are solely able to provide the energy requirement of GBM cells. Thus, based on our metabolomics data stated below we hypothesize that GBM cells adapt to chronic hypoxia utilizing alternate metabolic pathways such as peroxisomal fatty acid oxidation (FAO). Here we have explored the role of Acyl coenzyme A oxidase (ACOX-1), a rate limiting enzyme for peroxisomal FAO in the metabolic adaptations to hypoxia and as a potential drug target. Methods: U251 CRISPR dox inducible ACOX1 (KO) cells and patient derived primary GBM were grown under normoxic and hypoxic conditions (2%O2, 5%CO2, 70% humidity). Metabolic analysis was done using UHPLC-MS and LCMS analysis. RNAseq was done using TopHat2 (genome alignment), HTSeq, and DEseq to study the differential gene expression at cut-off of 2-fold change, and p<0.05. GSEA was used to interpret biological pathways. NCr/SCID (Severe Combined Immunodeficiency) mice (n=5) inoculated with U251 ACOX1 knockout cells expressing luciferase were used and treated with anti-angiogenic agent pazopanib at 30 mg/kg, 5 days a week. The U251 luciferase-labeled tumors were imaged starting weekly for tumor volumes using the IVIS (In Vivo Imaging System) Lumina. Results: In patient derived primary GBM cell lines alterations in metabolites in chronic hypoxia compared to normoxia, such as decrease in citric acid cycle metabolites, increase in glycolytic metabolites, and dysfunctional mitochondrial metabolites. In RNA seq downregulation of genes PCK1, SCD, SREBF1, and PIPOX (p<1x108) involved in PPAR and lipid metabolism pathways in hypoxia versus normoxia were noted. Inhibition of ACOX-1 displayed increased survival in mice administered (p=6.7E-04) with pazopanib compared to the U251 parental control group. Conclusion: There is metabolomic reprogramming in ACOX-1 KO cells channeled predominantly through FAO as seen through the downregulation of genes downstream of the PPAR signaling pathway, mediated through SREBF1 the master regulator of lipid metabolism. Thus, these genes could be future targets for therapy in combination with anti-angiogenic therapies that aggravate hypoxia in GBM. Citation Format: Ayon Bhattacharya, Laura Caflisch, Henriette Balinda, Alessia Lodi, Mei Zhou, Mengxing Li, Jennifer Chiou, Renu Pandey, Gangadhara R. Sareddy, Stefano Tiziani, Yidong Chen, Ratna K. Vadlamudi, Andrew J. Brenner. Metabolic adaptations of glioblastoma in hypoxia is mediated by acox-1 [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 4422.

Involvement of the tumour necrosis factor receptor system in glioblastoma cell death induced by palbociclib-heptamethine cyanine dye conjugate

Glioblastoma is the most common and aggressive primary brain tumour in adults. The development of anti-brain cancer agents are challenged by the blood-brain barrier and the resistance conferred by the local tumour microenvironment. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence compounds that have recently emerged as promising agents for drug delivery. We conjugated palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, to an HMCD, MHI-148, and conducted drug activity analysis on primary patient-derived glioblastoma cell lines. In addition to the expected cytostatic activity, our in vitro studies revealed that palbociclib-MHI-148 conjugate resulted in an almost 100-fold increase in cytotoxicity compared to palbociclib alone. This shift of palbociclib from cytostatic to cytotoxic when conjugated to MHI-148 was due to increased DNA damage, as indicated by an increase in γH2AX foci, followed by an increased expression of key extrinsic apoptosis genes, including TP53, TNFR1, TRAIL, FADD and caspase 8. In addition, we observed a time-dependent increase in the cell surface expression of TNFR1, consistent with an observed increase in the secretion TNFα, followed by TNFR1 endocytosis at 48 h. The treatment of patient GBM cells with the palbociclib-MHI-148 conjugate prevented TNFα-induced NFκB translocation, suggesting conjugate-induced TNFR1 signalling favoured the TNFR1-mediated apoptotic response rather than the pro-inflammatory response pathway. Notably, pharmacological inhibition of endocytosis of TNFR1, and siRNA-knockdown of TNFR1 reversed the palbociclib-MHI-148-induced cell death. These results show a novel susceptibility of glioblastoma cells to TNFR1-dependent apoptosis, dependent on inhibition of canonical NFκB signalling using our previously reported palbociclib-HMCD conjugate.9u5DmcuTyYJ7m7Hnyrq7sRVideo

Effect of Oxygen Tension on Glioblastoma Cell Growth

Background and Hypothesis:Glioblastoma (GBM) is an aggressive primary malignancy of the CNS with a dismal prognosis (~15-20 months) despite standard of care therapies. The poor prognosis of GBM despite ongoing research may be due to inaccuracies in preclinical models of the disease. Cell lines typically used to study GBM are exposed to ambient air containing 21% oxygen (normoxia compared to physiologic oxygen tension, physoxia, ~5%). In extracranial tumors, exposure to the oxygen in ambient air triggers epigenetic changes that alter cell growth, metabolism, and treatment responsiveness. The aim of this study is to obtain preliminary data on the impact ofphysiologic oxygen tensions on primary glioma cell growth in vitro.
 Experimental Design:Growth of primary glioma cell lines (GB43, GB10, GB001) and one immortalized cell line (293T HEK) was assessed in normoxia and physoxia. All cell lines were plated at 10,000 cells per well. Cells were harvested and counted in triplicate on days 2, 4, 6, 8, 10, 12. On each day the cells were counted, the media in the remaining wells was changed. This experiment was repeated three times. Wound healing assays with all cell lines were also performed at normoxia and physoxia.
 Results:Cells line growth curves were plotted and showed consistent exponential growth after counting on day two. From these graphs, the cell doubling time was calculated over a period of four days during which the cells were undergoing exponential growth.
 Conclusions:Current results indicated that primary glioblastoma cell lines grow at different rates at differing oxygen tensions. In ongoing studies, we are exploring the effect of low oxygen tensions on functional assays such as wound-healing. Future work will assess rates of growth and functional consequence of physoxia in tumor samples never exposed to ambient air to most accurately recapitulate the in-situ environment.

STEM-16. PROMOTING THE CANCER STEM CELL SUBPOPULATION AND PHENOTYPE IN GLIOBLASTOMA VIA EXTRACELLULAR VESICLE CROSS-TALK

Abstract INTRODUCTION Extracellular vesicles (EVs) released from cancer cells contain bioactive molecules that have been long implicated in tumorigenesis. In glioblastoma (GBM), there is mounting evidence that EVs play a role in critical processes including angiogenesis, immune suppression, and invasion. GBM secreted EVs have also been demonstrated to promote a cancer stem cell (CSC) phenotype by transporting stemness markers and proteins (I.e. Notch-1) involved in stem-cell activating pathways. Understanding the maintenance of CSCs, which contributes to tumor proliferation, treatment resistance, and re-expansion of the tumor bulk, can offer insight into new therapeutic drug targets that can disrupt this subpopulation. METHODS EVs were isolated from patient-derived primary GBM cell lines maintained as adherent cultures and free floating spheres (n = 3) using a staged ultracentrifugation technique. Normal astrocytes were used as the control group (n = 1). Protein expression profiles were then generated via high-throughput mass spectrometry for both EVs and whole cell lysate samples from the same cell line and compared against each other. RESULTS EGF-containing fibulin extracellular matrix protein 1 (EFEMP1) was shown to be upregulated in GBM derived EVs compared to control. Further analysis shows that EFEMP1 is enhanced in adherent cultures compared to free floating sphere cultures. CONCLUSION EFEMP1 is an extracellular matrix protein that has been identified as an oncogenic protein in GBM by activating Notch-1, Akt, and NF-Kb signaling pathways resulting in increased Temozolomide resistance, cellular proliferation, and invasion, all of which are hallmark characteristics of CSCs. EFEMP1 is also a downstream effector in the HIF-1 pathway, which has been strongly correlated in the maintenance of CSCs. Our results therefore demonstrate a possible EV mediated mechanism for the maintenance of the CSC population and phenotype in GBM.

Ribosomal S6 protein kinase 4 promotes resistance to EZH2 inhibitors in glioblastoma.

Glioblastoma (GBM) is a highly malignant type of brain tumor with limited treatment options. Recent research has focused on epigenetic regulatory factors, such as Enhancer of Zeste Homolog 2 (EZH2), which plays a role in gene expression through epigenetic modifications. EZH2 inhibitors have been developed as potential therapeutic agents for GBM, but resistance to these inhibitors remains a considerable challenge. This study aimed to investigate the role of ribosomal S6 protein kinase 4 (RSK4) in GBM and its association with resistance to EZH2 inhibitors. We first induced drug resistance in primary GBM cell lines by treatment with an EZH2 inhibitor and observed increases in the expression of stemness markers associated with glioblastoma stem cells (GSCs) in the drug-resistant cells. We also found high expression of RSK4 in GBM patient samples and identified the correlation of high RSK4 expression with poor prognosis and GSC marker expression. Further experiments showed that knocking down RSK4 in drug-resistant GBM cells restored their sensitivity to EZH2 inhibitors and decreased the expression of GSC markers, thus reducing their self-renewal capacity. From a mechanistic perspective, we discovered that RSK4 directly phosphorylates EZH2, activating the EZH2/STAT3 pathway and promoting resistance to EZH2 inhibitors in GBM. We also found that combining EZH2 inhibitors with an RSK4 inhibitor called BI-D1870 had better inhibitory effects on GBM occurrence and progression in both in vitro and in vivo experiments. In conclusion, this study demonstrates that RSK4 enhances cancer stemness and mediates resistance to EZH2 inhibitors in GBM. Combination treatment with EZH2 inhibitors and RSK4 inhibitors is a promising potential therapeutic strategy for GBM. Collectively, our results strongly demonstrate that RSK4 regulates the EZH2/STAT3 pathway to promote GSC maintenance and EZH2i resistance in a PRC2-independent manner, indicating that RSK4 is a promising therapeutic target for GBM.

Formulation of multicomponent inclusion complex of cyclodextrin-amino acid with Chrysin: Physicochemical characterization, cell viability and apoptosis assessment in human primary glioblastoma cell line

Chrysin (CR) is a water-insoluble drug reported for different therapeutic effects. The microwave irradiation method was used in this study to create a multicomponent inclusion complex (CR-MC) containing CR (drug) and carrier hydroxyl propyl beta cyclodextrin (HP β CD) and L-arginine (LA). The prepared inclusion complex (CR-MC) was evaluated for dissolution study and results were compared with chrysin physical mixture (CR-PM). Further, the samples were assessed for infra-red (IR), nuclear magnetic resonance (NMR), differential scanning calorimeter (DSC), scanning electron microscope (SEM) and molecular docking. Finally, the cell viability, reactive oxygen species and flow cytometer studies were also assessed to check the potential of the prepared inclusion complex on the human primary glioblastoma cell line (U87-MG cell). The phase solubility findings revealed a stability constant (773 mol L−1) as well as a complexation efficiency of 0.027. The dissolution study displayed a significant increase in CR release from CR-MC (99.03 ± 0.39%) > CR-PM (70.58 ± 1.16%) > pure CR (35.29 ± 1.55%). NMR and IR spectral data revealed no interaction between CR and carriers. SEM and DSC study results revealed the conversion into amorphous form. The molecular docking results illustrated a high docking score, which supports the findings of complex formation. The cell viability, reactive oxygen species, and flow cytometry studies results showed enhanced activity from CR-MC against the tested human primary glioblastoma cell line. From the results it has been observed that chrysin solubility significantly increased after complexation and there in vitro activity also enhanced against cancer cell line.

Aloe-Emodin Overcomes Anti-Cancer Drug Resistance to Temozolomide and Prevents Colony Formation and Migration in Primary Human Glioblastoma Cell Lines NULU and ZAR.

Glioblastoma, the most dangerous and aggressive type of CNS tumor, appears resistant to many chemotherapy drugs. In the patient-derived glioma cell lines NULU and ZAR, which exhibit drug-resistant phenotypes, we investigated the effect of combined AE (Aloe-emodin) and TMZ (temozolomide) and found a significant additive inhibitory effect on cell growth and a promising cytotoxic effect on both cell lines compared to treatment with single agents. We also examined the effect of combined AE and TMZ treatment on the drug-resistance protein MGMT. The results suggest that using AE combined with traditional drugs restores drug resistance in both primary resistant cell lines (NULU and ZAR). Furthermore, migration assays and scratch tests showed that the combined use of AE and TMZ can slow down the colony formation and migration of glioblastoma cells. These convincing results suggest that AE could be a natural adjuvant agent to potentiate the effects of traditional drugs (TMZ) and overcome drug resistance in glioblastoma cells.

A Potential Anti-Glioblastoma Compound LH20 Induces Apoptosis and Arrest of Human Glioblastoma Cells via CDK4/6 Inhibition.

Glioblastoma (GBM) is a deadly brain tumor characterized by signaling dysregulation and aberrant cell cycle control. The CDK4/6-Rb axis is dysregulated in approximately 80% of all GBM cases. In this study, the anti-GBM effect of a novel pyrimidin-2-amine, LH20 was evaluated in vitro using the primary GBM cell lines U87MG and U251. GBM cells were administered LH20 at concentrations of 0.1, 1, 4, 8, 10, 20, 100, and 200 µM for 24 and 48 h, and the proliferation rate was evaluated using a CCK8 assay. Migration, apoptosis, and cell cycle were also assessed using a wound healing assay, Annexin V-FITC/PI apoptosis assay, and cell cycle staining, respectively. The targets of LH20 were predicted using SwissTargetPrediction and molecular docking. Western blotting analysis was performed to confirm the anti-GBM mechanism of LH20. We found that at concentrations of 4, 8, and 10 µM, LH20 significantly inhibited the proliferation and migration of U87MG and U251 cells, induced late phase apoptosis, promoted tumor cell necrosis, and arrested the G2/M phase of the cell cycle. LH20 also inhibited CDK4 and CDK6 activities by decreasing the phosphorylation of Rb. Our results suggest LH20 as a potential treatment strategy against GBM.

Abstract 6185: Debio 0123 is a selective WEE1 inhibitor that effectively penetrates the brain and demonstrates anti-tumor activity in preclinical models of glioblastoma

Abstract Background: Glioblastoma (GBM) is one of the most aggressive and hard-to-treat cancers with a 5-year survival rate of 5.8%, in part due to the presence of the blood brain barrier (BBB) that prevents most therapeutics from reaching the tumors at efficacious concentrations. Debio 0123 is a WEE1 inhibitor that has previously shown significant preclinical efficacy in combination with DNA damaging agents or radiotherapy and is currently being investigated in phase 1 clinical studies either as a monotherapy or in combination with carboplatin. Herein we present data that demonstrates Debio 0123 effectively penetrates in to the brain and subsequently leads to anti-tumor activity in GBM models both in vitro and in vivo as a monotherapy or in combination with standard-of-care therapies (SOC). Methods: Radio- and chemo-sensitizing effects of Debio 0123 were evaluated in GBM cell lines in vitro using standard clonogenic assays or cytotoxicity assays. In vivo, distribution and bioanalysis studies were performed to determine the amount of Debio 0123 present in the different parts of the brain and intracranially implanted tumors. Efficacy of Debio 0123 as monotherapy or in combination with the SOC, temozolomide (TMZ), was further assessed in vivo in U87MG-luc intracranial or subcutaneous xenograft models. Results: In vitro, Debio 0123 in combination with radiation enhanced cell death in GBM cell lines and reduced TMZ IC50 in primary GBM cell lines. In vivo studies demonstrated substantial Debio 0123 brain penetration in mice, rats and monkeys, with mean brain-to-plasma concentration ratios of ~0.6 and 1.52 and 4, respectively. Furthermore, a mean tumor-to-plasma ratio of 0.62 was determined in orthotopically implanted brain tumors in mice. In efficacy studies, Debio 0123 administered at 30 or 60mg/kg every day for 28 days displayed significant anti-tumor activity in a GBM xenograft model U87-MG implanted in immunodeficient mice either subcutaneously, with up to 57.5% tumor growth inhibition, or orthotopically in the brain, with up to 73.7% tumor growth inhibition. Finally, efficacy of Debio 0123 in combination with TMZ was also assessed in intracranially transplanted U87-MG tumors. Here, TMZ was highly active, however, the combination of Debio 0123 and TMZ resulted in significantly enhanced anti-tumor activity leading to sustained complete regressions in 75% of animals for up to 100 days and was well tolerated throughout the 28-day treatment. Conclusion: These results demonstrate that following oral administration, Debio 0123 effectively crosses the BBB, has significant efficacy as a monotherapy and improves response to standard-of-care chemo or radiotherapy in preclinical models. Collectively, these data highlight Debio 0123 as a promising candidate for further clinical development in GBM patients that may provide improved response to SOC therapy. Citation Format: Luke Piggott, Noemie Luong, Frederic Massiere, Annett Kunze, Christophe Chardonnens, Anne Vaslin. Debio 0123 is a selective WEE1 inhibitor that effectively penetrates the brain and demonstrates anti-tumor activity in preclinical models of glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6185.

STEM-05. GLIOBLASTOMA NEUROSPHERES DISPLAY STEMNESS CHARACTERISTICS AND UPREGULATION OF THE VEGF-A PATHWAY

Abstract INTRODUCTION Glioma stem cells (GSC) constitute a subpopulation of tumor cells with stem-cell like properties and are believed to enhance glioblastoma’s ability to grow and confer resistance against chemoradiotherapy. It has been shown that non-adherent three-dimensional (3D) growth conditions facilitate neurosphere formation and enable isolation of GSCs in vitro. This can serve as an invaluable tool to better understand the role of GSCs in glioblastoma pathogenesis. OBJECTIVE We aimed to evaluate differentially expressed genes in non-adherent versus adherent conditions using primary tumor glioblastoma cell lines; and to identify alterations in various signaling pathways that are paramount to their growth and maintenance. Furthermore, we targeted the EMP2-HIF1α axis, a proposed regulator of VEGF expression, to determine its effects on cell viability. METHODS Three primary tumor glioblastoma cell lines were generated and cultured under adherent or non-adherent growth conditions. Sequencing was performed on the Illumina HiSeq3000 system, and gene counts were normalized by TMM. A pair-wise statistical analysis of over 20,000 genes was performed between the two conditions. A p-value < 0.05 was considered statistically significant. RESULTS After 72 hours of growth under 3D conditions, primary glioblastoma cells show an increase in stem-like properties, measured via an upregulation of SOX2 and nestin. RNA sequencing demonstrated a significant increase in expression of the following transcripts in 3D conditions compared to those grown on adherent plates: VEGF-A, EGFR, PDGFR-A, FGFR3, and TGFB2. Treatment with anti-EMP2 monoclonal antibodies significantly reduced cellular proliferation. CONCLUSION Our results demonstrate that neurospheres upregulate growth factor pathways to enable their survival in 3D conditions, suggesting an inherently different genetic landscape between differentiated tumor cells and GSCs. Future studies will investigate the cellular mechanisms responsible for upregulation of these genes; and to ultimately design more effective targeted therapeutic agents.

Establishment and characteristics of GWH04, a new primary human glioblastoma cell line.

Glioblastoma multiforme (GBM) is a common and fatal disease of the central nervous system. GBM cell lines are fundamental tools used in GBM research. The establishment of novel continuous GBM cell lines with clear genetic backgrounds could facilitate the exploration of molecular mechanisms and the screening and evaluation of antitumor drugs in GBM studies. In the present study, a novel primary glioblastoma cell line was established, named GWH04, from a patient with GBM, and its STR genotype and various tumor parameters were examined. The STR information of GWH04 was identical to that of the original primary tumor tissue. Compared with existing cell lines, GWH04 had a similar in vitro proliferation rate as the U87 cell line, but a faster rate than the GL15 cell line, and substantial soft agar clone-formation capacity and subcutaneous and intracranial tumorigenic capacity. For drug sensitivity test, half maximal inhibitory concentration assays for multiple drugs were performed in these three cell lines, and GWH04 cells were found to be resistant to temozolomide. Aneuploid karyotype with a median of 84 chromosomes was possessed by GWH04, as well as chromosomal structural abnormalities, such as broken chromosomes, double centromere chromosomes, homogeneous staining regions, and double microbodies. Gene sequencing further revealed the mutational status of genes TP53, PTEN, PDGFRA, ERBB2, BRCA1, NF1, and MLH1 and the promoter region of telomerase reverse transcriptase (C228T) in this cell line. Altogether, these results indicated that GWH04 will be a useful tool for human GBM studies both in vitro and in vivo.

The role of Shikonin in improving 5-aminolevulinic acid-based photodynamic therapy and chemotherapy on glioblastoma stem cells

Glioblastoma multiforme is a malignant neoplasia with a median survival of less than two years and without satisfactory therapeutic options. The so-called glioblastoma stem cells escape the established radio- and chemotherapies and lead to tumor recurrence in most cases. The alkaloid Shikonin with its various anti stem cell properties and the interstitial photodynamic therapy with 5-aminolevulinic acid seem to be promising new options in the therapy of glioblastoma. In this study, in vitro investigations were performed to observe the influence of Shikonin on viability, proliferation, induction of apoptosis and the capability of forming tumor spheres in U-87 MG and the primary glioblastoma cell line GB14. The combined effect with the chemotherapeutic temozolomide and photodynamic treatment on the mRNA expression of glioma specific stem cell markers and further examined intracellular protoporphyrin IX accumulation under Shikonin treatment was analyzed. Shikonin effectively inhibited the capability of forming tumor spheres and enhanced temozolomide effectiveness in the reduction of proliferation and in the induction of apoptosis. Additionally, Shikonin increased the mRNA expression of the tumor suppressing Neurofibromatosis type 1 (NF1) gene and showed modulating effects on intracellular protoporphyrin IX.

Abstract 1822: Selective inhibition of ZCCHC11/ZCCHC6 TUTases with genetic and pharmacological tools supports a role in glioblastoma cell growth

Abstract The purpose of the study was to determine if genetic and pharmacological inhibition of terminal uridyltransferases (TUTases) ZCCHC11 and ZCCHC6 could modulate Glioblastoma (GBM) cell growth in vitro. ZCCHC11 (also known as TUT4 and TENT3A) and ZCCHC6 (also known as TUT7 and TENT3B) catalyze uridylation of diverse RNA species. Recent evidence indicates that genetic perturbation of ZCCHC11/6 expression can disrupt cell proliferation of both immortalized and patient-derived primary GBM cell lines (Kim et al., Mol Cell 2020). We confirmed that genetic inhibition of ZCCHC11/6 with siRNA or CRISPR can decrease viability of U-87 MG and A-172 GBM cell lines, and we identified DK-MG as another GBM cell line with sensitivity to ZCCHC11/6 knockdown. Herein, we report the first novel, potent, and selective inhibitor of ZCCHC11/6, TS-1. Biochemically, TS-1 can block ZCCHC11/6-mediated uridylation of an RNA substrate in vitro (IC50 = 0.65 nM for recombinant ZCCHC11 protein; IC50 = 9.6 nM for recombinant ZCCHC6 protein). ZCCHC11/6 inhibitor TS-1 could decrease cell viability in U-87 MG, A-172 and DK-MG cells (cell viability IC50s ranging between 0.7-1.5 μM) and induce apoptosis and cell cycle arrest. A cellular assay detecting uridylation of miR-191 as a marker for ZCCHC11/6 activity was developed and validated. TS-1 strongly decreased uridylation of miR-191 in U-87 MG, A-172 and DK-MG cells (IC50 ranging between 10-40 nM). In contrast, TS-2, a weakly active enantiomer of TS-1 (enzyme IC50 = 0.15 μM and 1.8 μM against ZCCHC11 and ZCCHC6, respectively), had less impact on cell viability, apoptosis, cell cycle and uridylation in cells. In conclusion, we have verified ZCCHC11/6-dependency in a set of GBM cell lines and we have also developed a first-in class potent and selective small molecule that reduces in vitro Glioblastoma cell proliferation through selective inhibition of TUTases activity. Citation Format: Robinson Triboulet, Khikmet Sadykov, Jessica L. Johnson, Andrew R. Snyder, Sarah K. Knutson, Pavan Kumar, Christopher B. Mayo, Dillon Hawley, Andrew Madanjian, Ross L. Stein, David M. Wilson, Darren M. Harvey, Shomir Ghosh, Robert M. Campbell. Selective inhibition of ZCCHC11/ZCCHC6 TUTases with genetic and pharmacological tools supports a role in glioblastoma cell growth [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 1822.

Resibufogenin Targets the ATP1A1 Signaling Cascade to Induce G2/M Phase Arrest and Inhibit Invasion in Glioma.

Resibufogenin (RB) is a major active ingredient in the traditional Chinese medicine Chansu and has garnered considerable attention for its efficacy in the treatment of cancer. However, the anticancer effects and underlying mechanisms of RB on glioblastoma (GBM) remain unknown. Here, we found that RB induced G2/M phase arrest and inhibited invasion in a primary GBM cell line, P3#GBM, and two GBM cell lines, U251 and A172. Subsequently, we demonstrated that RB-induced G2/M phase arrest occurred through downregulation of CDC25C and upregulation of p21, which was caused by activation of the MAPK/ERK pathway, and that RB inhibited GBM invasion by elevating intercellular Ca2+ to suppress the Src/FAK/Paxillin focal adhesion pathway. Intriguingly, we confirmed that upon RB binding to ATP1A1, Na+-K+-ATPase was activated as a receptor and then triggered the intracellular MAPK/ERK pathway and Ca2+-mediated Src/FAK/Paxillin focal adhesion pathway, which led to G2/M phase arrest and inhibited the invasion of GBM cells. Taken together, our findings reveal the antitumor mechanism of RB by targeting the ATP1A1 signaling cascade and two key signaling pathways and highlight the potential of RB as a new class of promising anticancer agents.

Cucurbitacin B and Evodiamine imparts anti‐neoplastic effects against Glioblastoma Multiforme via PI3‐K/AKT signaling pathway

Glioblastoma Multiforme (GBM) is a highly malignant and aggressive brain tumor. Patients’ survival is around 6 to 12 months even with chemotherapy and surgery. Temozolomide is a first line chemotherapeutic treatment prescribed along with surgery but due to the high resistance to the drug and a low patient survival rate, new drugs and drug combinations must continue to be investigated and developed. Evodiamine, a marketed supplement, and Cucurbitacin B, a compound obtained from natural plants, have been identified as promising candidates to treat various neoplasms but has not been well studied in GBM, and especially in combination. The present study investigated the anti‐proliferating effects of Evodiamine (Evo) and Cucurbitacin‐B (CuB) individually and in combination in U‐87MG cell line (Human Primary Glioblastoma cell line) and elucidated probable mechanisms of action via PI3K/AKT pathway in GBM. Results demonstrated that both CuB and Evo individually decreased the U‐87MG cell viability in a dose dependent manner. The effect was pronounced with drug combination. CuB and Evo imparted their effect via the PI3K/P‐AKT signaling pathway. In addition, Cells treated with CuB and Evo showed a decrease in Bcl2, an anti‐apoptotic factor, and an increase in Bax, a pro‐apoptotic factor, expression; a hallmark of induction of apoptosis. The increased expression of Cleaved Caspase‐3 and p‐c Jun have also been observed. Overall, Cucurbitacin and evodiamine individually and especially in combination proved to be potential anti‐neoplastic agents in U‐87MG cells and may be considered as potential adjuvant therapies for GBM.

Ageritin-The Ribotoxin-like Protein from Poplar Mushroom (Cyclocybe aegerita) Sensitizes Primary Glioblastoma Cells to Conventional Temozolomide Chemotherapy.

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.