Glioblastoma Multiforme Cell Lines Research Articles

Investigating the Effects of Olaparib on the Susceptibility of Glioblastoma Multiforme Tumour Cells to Natural Killer Cell-Mediated Responses.

Glioblastoma multiforme (GBM) is the most common adult primary brain malignancy, with dismal survival rates of ~14.6 months. The current standard-of-care consists of surgical resection and chemoradiotherapy, however the treatment response is limited by factors such as tumour heterogeneity, treatment resistance, the blood-brain barrier, and immunosuppression. Several immunotherapies have undergone clinical development for GBM but demonstrated inadequate efficacy, yet future combinatorial approaches are likely to hold more promise. Olaparib is FDA-approved for BRCA-mutated advanced ovarian and breast cancer, and clinical studies have revealed its utility as a safe and efficacious radio- and chemo-sensitiser in GBM. The ability of Olaparib to enhance natural killer (NK) cell-mediated responses has been reported in prostate, breast, and lung cancer. This study examined its potential combination with NK cell therapies in GBM by firstly investigating the susceptibility of the GBM cell line T98G to NK cells and, secondly, examining whether Olaparib can sensitise T98G cells to NK cell-mediated responses. Here, we characterise the NK receptor ligand profile of T98G cells and demonstrate that Olaparib does not dampen T98G susceptibility to NK cells or elicit immunomodulatory effects on the function of NK cells. This study provides novel insights into the potential combination of Olaparib with NK cell therapies for GBM.

The FOXO1 inhibitor AS1842856 triggers apoptosis in glioblastoma multiforme and basal-like breast cancer cells.

Basal-like breast cancer (BBC) and glioblastoma multiforme (GBM) are poor-prognosis cancers that lack effective targeted therapies and harbor embryonic stem gene expression signatures. Recently, our group and others found that forkhead box transcription factor FOXO1 promotes stem gene expression in BBC and GBM cell lines. Given the critical role of cancer stem cells in promoting cancer progression, we examined the impact of FOXO1 inhibition with AS1842856 (a cell-permeable small molecule that directly binds to unphosphorylated FOXO1 protein to block transcriptional regulation) on BBC and GBM cell viability. We treated a set of BBC and GBM cancer cell lines with increasing concentrations of AS1842856 and found reduced colony formation. Treatment of BBC and GBM cancer cells with AS1842856 led to increases in FAS (FAS cell surface death receptor) and BIM (BCL2L11) gene expression, as well as increased positivity for markers for apoptosis such as annexin V and propidium iodide. Treatment with another FOXO1 inhibitor AS1708727 or FOXO1 RNAi also led to FAS induction. This work is the first to show that targeting BBC and GBM with FOXO1 inhibition leads to apoptosis. These novel findings may ultimately expand the repertoire of therapies for poor-prognosis cancers.

ProNGF Expression and Targeting in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most lethal adult brain cancer. Temozolomide (TMZ), the standard chemotherapeutic drug used in GBM, has limited benefit and alternate therapies are needed to improve GBM treatment. Nerve growth factor (NGF) and its precursor proNGF are increasingly recognized as stimulators of human tumor progression. The expression and stimulatory effect of NGF on GBM cell growth has previously been reported, but the status of proNGF in GBM is unreported. In this study, we have investigated proNGF expression and biological activity in GBM. A clinical cohort of GBM (n = 72) and low-grade glioma (n = 20) was analyzed by immunohistochemistry for proNGF and digital quantification. ProNGF expression was significantly increased in GBM compared to low grade gliomas and proNGF was also detected in patient plasma samples. ProNGF was also detected in most GBM cell lines by Western blotting. Although anti-proNGF blocking antibodies inhibited cell growth in GBM cells with methylated MGMT gene promoter, targeting proNGF could not potentiate the efficacy of TMZ. In subcutaneous xenograft of human GBM cells, anti-proNGF antibodies slightly reduced tumor volume but had no impact on TMZ efficacy. In conclusion, this data reveals that proNGF is overexpressed in GBM and can stimulate cancer cell growth. The potential of proNGF as a clinical biomarker and therapeutic target warrants further investigations.

Polyploid/Multinucleated Giant and Slow-Cycling Cancer Cell Enrichment in Response to X-ray Irradiation of Human Glioblastoma Multiforme Cells Differing in Radioresistance and TP53/PTEN Status.

Radioresistance compromises the efficacy of radiotherapy for glioblastoma multiforme (GBM), the most devastating and common brain tumor. The present study investigated the relationship between radiation tolerance and formation of polyploid/multinucleated giant (PGCC/MGCC) and quiescent/senescent slow-cycling cancer cells in human U-87, LN-229, and U-251 cell lines differing in TP53/PTEN status and radioresistance. We found significant enrichment in MGCC populations of U-87 and LN-229 cell lines, and generation of numerous small mononuclear (called Raju cells, or RJ cells) U-87-derived cells that eventually form cell colonies, in a process termed neosis, in response to X-ray irradiation (IR) at single acute therapeutic doses of 2-6 Gy. For the first time, single-cell high-content imaging and analysis of Ki-67- and EdU-coupled fluorescence demonstrated that the IR exposure dose-dependently augments two distinct GBM cell populations. Bifurcation of Ki-67 staining suggests fast-cycling and slow-cycling populations with a normal-sized nuclear area, and with an enlarged nuclear area, including one resembling the size of PGCC/MGCCs, that likely underlie the highest radioresistance and propensity for repopulation of U-87 cells. Proliferative activity and anchorage-independent survival of GBM cell lines seem to be related to neosis, low level of apoptosis, fraction of prematurely stress-induced senescent MGCCs, and the expression of p63 and p73, members of p53 family transcription factors, but not to the mutant p53. Collectively, our data support the importance of the TP53wt/PTENmut genotype for the maintenance of cycling radioresistant U-87 cells to produce a significant amount of senescent MGCCs as an IR stress-induced adaptation response to therapeutic irradiation doses.

Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines

Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.

Effect of Copper Chelators via the TGF-β Signaling Pathway on Glioblastoma Cell Invasion.

Glioblastoma multiforme (GBM) is a fast-growing and aggressive type of brain cancer. Unlike normal brain cells, GBM cells exhibit epithelial-mesenchymal transition (EMT), which is a crucial biological process in embryonic development and cell metastasis, and are highly invasive. Copper reportedly plays a critical role in the progression of a variety of cancers, including brain, breast, and lung cancers. However, excessive copper is toxic to cells. D-penicillamine (DPA) and triethylenetetramine (TETA) are well-known copper chelators and are the mainstay of treatment for copper-associated diseases. Following treatment with copper sulfate and DPA, GBM cells showed inhibition of proliferation and suppression of EMT properties, including reduced expression levels of N-cadherin, E-cadherin, and Zeb, which are cell markers associated with EMT. In contrast, treatment with copper sulfate and TETA yielded the opposite effects in GBM. Genes, including TGF-β, are associated with an increase in copper levels, implying their role in EMT. To analyze the invasion and spread of GBM, we used zebrafish embryos xenografted with the GBM cell line U87. The invasion of GBM cells into zebrafish embryos was markedly inhibited by copper treatment with DPA. Our findings suggest that treatment with copper and DPA inhibits proliferation and EMT through a mechanism involving TGF-β/Smad signaling in GBM. Therefore, DPA, but not TETA, could be used as adjuvant therapy for GBM with high copper concentrations.

Modulatory activity of ADNP on the hypoxia‑induced angiogenic process in glioblastoma.

Glioblastoma multiforme (GBM) is a brain cancer with a poor prognosis that affects adults. This is a solid tumor characterized by a high rate of cell migration and invasion. The uncontrolled cell proliferation creates hypoxic niches in the tumor mass, which leads to the overexpression of hypoxia‑inducible factors (HIFs). This induces the activation of the vascular endothelial growth factor (VEGF), which is responsible for uncontrolled neoangiogenesis. Recent studies have demonstrated the anti‑invasive effect of pituitary adenylate cyclase‑activating peptide (PACAP) in GBM. PACAP effects on the central nervous system are also mediated through the activity‑dependent neuroprotective protein (ADNP) activation. To date, no evidence exists regarding its role in GBM. Therefore, the ADNP involvement in GBM was investigated. By analyzing ADNP expression in a human GBM sample through confocal microscopy, a high ADNP immunoreactivity was detected in most glial cells and its predominant expression in hypoxic areas overexpressing HIF‑1α was highlighted. To investigate the role of ADNP on the HIF‑VEGF axis in GBM, a human U87MG GBM cell line was cultured with a hypoxic mimetic agent, deferoxamine, and cells were treated with the smallest active fragment of ADNP, known as NAP. The protein expression and distribution of HIF‑1α and VEGF was detected using western blot analysis and immunofluorescence assay. Results demonstrated that ADNP modulates the hypoxic‑angiogenic pathway in GBM cells by reducing VEGF secretion, detected through ELISA assay, as well as modulating their migration, assessed through wound healing assay. Although deeper investigation is necessary, the present study suggested that ADNP could be involved in PACAP anti‑invasive effects in GBM.

SREBP2/Rab11s/GLUT1/6 network regulates proliferation and migration of glioblastoma.

Cholesterol serves a vital role in the occurrence and development of glioblastoma multiforme (GBM). Furthermore, cholesterol synthesis is regulated by sterol regulatory element-binding protein 2 (SREBP2), and certain glucose transporters (GLUTs) and Ras-related protein Rab11 (Rab11) small GTPase family members (Rab11s) may contribute to the process. The Cancer Genome Atlas was used to analyze the relationship between prognosis and GLUT gene expressions. To investigate the regulatory effect of Rab11s and SREBP2 on GLUTs during tumor progression, single cell RNA sequencing (scRNA-seq), western blotting and reverse transcription-quantitative PCR were performed on glioma tissues and the T98G GBM cell line. Cell viability and migration were assessed by performing MTT and wound healing assays, respectively. Moreover, the dual-luciferase reporter gene assay was conducted to predict the sterol regulatory elements in the promoter regions of the target genes. The results demonstrated that high SREBP2, GLUT1 and GLUT6 expression was associated with poor survival of patients with GBM. ScRNA-seq distinguished glioblastoma cells by EGFR and indicated the related lipid metabolism signaling pathways. Moreover, the results indicated that GLUT1 and GLUT6 were regulated by SREBP2 and Rab11s. Rab11s and SREBP2 also contributed to T98G cell viability and migration. Additionally, the results indicated that Rab11s, GLUT1 and GLUT6 were transcriptionally regulated by SREBP2. Therefore, the present study suggested that the SREBP2/Rab11/GLUT network promoted T98G cell growth, thus, identifying potential therapeutic targets for GBM.

Inhibition of MZF1/c-MYC Axis by Cantharidin Impairs Cell Proliferation in Glioblastoma

Myeloid zinc finger 1 (MZF1), also known as zinc finger protein 42, is a zinc finger transcription factor, belonging to the Krüppel-like family that has been implicated in several types of malignancies, including glioblastoma multiforme (GBM). MZF1 is reportedly an oncogenic gene that promotes tumor progression. Moreover, higher expression of MZF1 has been associated with a worse overall survival rate among patients with GBM. Thus, MZF1 may be a promising target for therapeutic interventions. Cantharidin (CTD) has been traditionally used in Chinese medicine to induce apoptosis and inhibit cancer cell proliferation; however, the mechanism by which CTD inhibits cell proliferation remains unclear. In this study, we found that the expression of MZF1 was higher in GBM tissues than in adjacent normal tissues and low-grade gliomas. Additionally, the patient-derived GBM cells and GBM cell lines presented higher levels of MZF1 than normal human astrocytes. We demonstrated that CTD had greater anti-proliferative effects on GBM than a derivative of CTD, norcantharidin (NCTD). MZF1 expression was strongly suppressed by CTD treatment. Furthermore, MZF1 enhanced the proliferation of GBM cells and upregulated the expression of c-MYC, whereas these effects were reversed by CTD treatment. The results of our study suggest that CTD may be a promising therapeutic agent for patients with GBM and suggest a promising direction for further investigation.

Saikosaponin D Inducing Apoptosis and Autophagy through the Activation of Endoplasmic Reticulum Stress in Glioblastoma.

Saikosaponin D (SSD), a saponin derivative, is extracted from Bupleurum falcatum. It exhibits an inhibitory effect on a number of tumor cells and is relatively safe when used at therapeutic doses. However, its effects on glioblastoma multiforme (GBM) have not been fully explored. This study is aimed at investigating the cytotoxic effects of SSD in GBM cell lines. SSD induces apoptosis and autophagy by activating endoplasmic reticulum (ER) stress in GBM cells. GBM cell proliferation activity and morphology were observed using the Cell Counting Kit-8 assay and hematoxylin and eosin staining. Hoechst 33258 fluorescence staining and flow cytometry were performed to assess apoptosis. Western blotting and immunocytochemical staining were used to detect protein expression and distribution. SSD significantly inhibited the proliferation of RG-2, U87-MG, and U251 cells in a dose-dependent manner, and the proportion of apoptotic cells increased significantly. Additionally, the expressions of ER-, apoptosis-, and autophagy-related proteins were significantly upregulated and distributed in the cytoplasm and nucleus. Therefore, SSD may be considered a novel treatment option for GBM. This study demonstrated the anti-GBM effect of SSD from the perspectives of cell apoptosis and autophagy.

Guggulsterone inhibits migration and invasion through proteasomal and lysosomal degradation in human glioblastoma cells

Glioblastoma multiforme (GBM) is a deadly brain malignancy, and current therapies offer limited survival benefit. The phytosterol guggulsterone (GS) has been shown to exhibit antitumor efficacy. This study aimed to investigate the effects of GS on migration and invasion and its underlying mechanisms in human GBM cell lines. After GS treatment, the survival rate of GBM cells was reduced, and the migration and invasion abilities of GBM cells were significantly decreased. There was also concomitant decreased expression of focal adhesion complex, matrix metalloproteinase-2 (MMP2), MMP9 and cathepsin B. Furthermore, GS induced ERK phosphorylation and autophagy, with increased p62 and LC3B-II expression. Notably, treatment of in GBM cells with the proteasome inhibitor MG132 or the lysosome inhibitor NH4Cl reversed the GS-mediated inhibition of migration and invasion. In an orthotopic xenograft mouse model, immunohistochemical staining of brain tumor tissues demonstrated that MMP2 and cathepsin B expression was reduced in GS-treated mice. GS treatment inhibited GBM cell migration and invasion via proteasomal and lysosomal degradation, suggesting its therapeutic potential in clinical use in the future.

Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration.

(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.

MCM4 is a novel prognostic biomarker and promotes cancer cell growth in glioma

Gliomas account for 75% of all primary malignant brain tumors in adults and result in high mortality. Accumulated evidence has declared the minichromosome maintenance protein complex (MCM) gene family plays a critical role in modulating the cell cycle and DNA replication stress. However, the biological function and clinic characterization of nine MCM members in low-grade glioma are not yet clarified. In this study, we utilized diverse public databases, including The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), Rembrandt, Human Protein Atlas (HPA), Linkedomics, cbioportal, Tumor and Immune System Interaction Database (TISIDB), single-sample GSEA (ssGSEA), Tumor Immune Estimation Resource (TIMER), Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Therapeutics Response Portal databases to explore the mRNA and protein expression profiles, gene mutation, clinical features, diagnosis, prognosis, signaling pathway, tumor mutational burden (TMB), immune subtype, immune cell infiltration, immune modulator and drug sensitivity of nine MCMs. Afterward, qRT-PCR was utilized to detect the expression of the MCM family in glioblastoma multiforme (GBM) cell lines. The one-, three-, or five-year survival rate was predicted by utilizing a nomogram established by cox proportional hazard regression. In this study, we found that nine MCMs were consistently up-regulated in glioma tissues and glioma cell lines. Elevated nine MCMs expressions were significantly correlated with a higher tumor stage, isocitrate dehydrogenase (IDH) mutates, 1p/19q codeletion, histological type, and primary therapy outcome. Survival analyses showed that higher expression of MCM2-MCM8 (minichromosome maintenance protein2-8) and MCM10 (minichromosome maintenance protein 10) were linked with poor overall survival (OS) and progression-free survival (PFS) in glioma patients. On the other hand, up-regulated MCM2-MCM8 and MCM10 were significantly associated with shorter disease-specific survival (DSS) in glioma patients. Univariate and multivariate analyses revealed that MCM2 (minichromosome maintenance protein2), MCM4 (minichromosome maintenance protein 4), MCM6 (minichromosome maintenance protein 6), MCM7 (minichromosome maintenance protein 7) expression and tumor grade, 1p/19q codeletion, age, and primary therapy outcome were independent factors correlated with the clinical outcome of glioma patients. More importantly, a prognostic MCMs model constructed using the above five prognostic genes could predict the overall survival of glioma patients with medium-to-high accuracy. Furthermore, functional enrichment analysis indicated that MCMs principal participated in regulating cell cycle and DNA replication. DNA copy number variation (CNV) and DNA methylation significantly affect the expression of MCMs. Finally, we uncover that MCMs expression is highly correlated with immune cell infiltration, immune modulator, TMB, and drug sensitivity. In summary, this finding confirmed that MCM4 is a potential target of precision therapy for patients with glioma.

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.

The IAP antagonist birinapant enhances chimeric antigen receptor T cell therapy for glioblastoma by overcoming antigen heterogeneity

Antigen heterogeneity that results in tumor antigenic escape is one of the major obstacles to successful chimeric antigen receptor (CAR) Tcell therapies in solid tumors including glioblastoma multiforme (GBM). To address this issue and improve the efficacy of CAR Tcell therapy for GBM, we developed an approach that combines CAR Tcells with inhibitor of apoptosis protein (IAP) antagonists, a new class of small molecules that mediate the degradation of IAPs, to treat GBM. Here, we demonstrated that the IAP antagonist birinapant could sensitize GBM cell lines and patient-derived primary GBM organoids to apoptosis induced by CAR Tcell-derived cytokines, such as tumor necrosis factor. Therefore, birinapant could enhance CAR Tcell-mediated bystander death of antigen-negative GBM cells, thus preventing tumor antigenic escape in antigen-heterogeneous tumor models invitro and invivo. In addition, birinapant could promote the activation of NF-κB signaling pathways in antigen-stimulated CAR Tcells, and with a birinapant-resistant tumor model we showed that birinapant had no deleterious effect on CAR Tcell functions invitro and invivo. Overall, we demonstrated the potential of combining the IAP antagonist birinapant with CAR Tcells as a novel and feasible approach to overcoming tumor antigen heterogeneity and enhancing CAR Tcell therapy forGBM.

DDDR-40. OVERCOMING DRUG RESISTANCE BY TARGETING AUTOPHAGY REGULATED BY A LONG INTERGENIC NONCODING RNA

Abstract BACKGROUND Autophagy is a self-eating process that supports cancer cell survival. Autophagy activation during drug treatment often prolongs cancerous cell survival, causing drug resistance, as seen in glioblastoma multiforme (GBM) to temozolomide (TMZ). Thus, blocking autophagy is an appealing approach to overcoming cancer drug resistance. Long noncoding RNAs (LincRNAs) have emerged as potential targets. LincRNA LINC00467 (renamed ARLINC) was recently identified as a potential autophagy inhibitor in cancer cells. However, ARLINC’s mechanism of inhibition and whether its modulation can suppress autophagy in cancer remains unknown. OBJECTIVE Research Question: Can blocking autophagy restore the sensitivity of cancer cells to chemotherapy? Hypotheses: 1) ARLINC regulates chemotherapy drug sensitivity by inducing autophagy. 2) Overexpression of ARLINC can increase chemotherapy drug sensitivity, as measured by cell viability and autophagy assays METHODS Aim 1) Endogenous ARLINC levels in GBM cell lines will be measured using qRT-PCR. Aim 2) Cell viability of GBM upregulated with pBabe-ARLINC (experiment) and pBabe (control) then exposed to increasing concentrations of TMZ will be analyzed using MTS assay. Student’s T-test will be used to determine significance. Aim 3) Autophagy flux will be measured in GBM lines that are transiently transfected with ARLINC compared to the empty vector via Cyto-ID assay. RESULTS Aim 1: Compared to Normal Human Astrocytes (NHA), U87MG had higher levels, LN18 and SF295 and lower, and LN229 had around the same levels of ARLINC. Aim 2: MTS cell viability assays in SF295 upregulated with ARLINC showed no significant decrease in TMZ sensitivity. Aim 3: Cyto-ID in U87MG and LN229 upregulated with ARLINC showed no significant decrease in autophagy flux. CONCLUSIONS/FUTURE WORK Results show no effect of ARLINC upregulation on cell viability and autophagy flux. Future work involves knockdown experiments to decrease intracellular levels of ARLINC and determine the effects of upregulation of autophagy on sensitization.

DDEL-08. NOVEL COMBINATION THERAPY USING PI3KINASE INHIBITION AND IMMUNE BASED THERAPY FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Abstract Hurdles to effective treatments for glioblastoma multiforme (GBM), the most aggressive form of brain cancer, are resistance to chemotherapy, radiation, and immune checkpoint inhibitors. GBMs, like other “difficult to treat” tumors, often over-utilize the phosphatidyl inositol 3 kinase (PI3K) pathway for survival, cell growth, and cell division, conferring both chemotherapy and radiation resistance. Furthermore, the PI3K pathway has been shown to modulate expression of Major Histocompatibility Complex (MHC) molecules, key components of immune recognition. Based on these observations, we examined the potential of a novel, small molecule PI3K inhibitor GCT.Glio.1 (NPT520-337), which was designed to cross the blood brain barrier, to increase the sensitivity of GBM to immune based therapies and radiation. We hypothesized that targeting PI3K would result in growth arrest and increase cell surface expression of MHC molecules and the PD-L1 checkpoint target. Treatment of GL261 mouse and human U251 GBM cell lines with GCT.Glio.1 caused growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1 and MHC class II, and synergized with radiation to cause growth arrest and cell death. Based on these results we designed and performed in vivo animal studies, implanting luciferase-flagged GL261 into the caudate nucleus of C57BL6 mice. We treated the animals orally with GCT.Glio.1 followed 24 hours later by anti-PD1 therapy in repeating cycles. The treatments significantly delayed progression of the tumor when compared to no treatment or either treatment alone, with several animals exhibiting complete regression as measured by MRI and bioluminescence. In conclusion, these results indicate that GCT.Glio.1 may sensitize GBM to immune checkpoint inhibitor and/or radiation therapies. Based on our encouraging pre-clinical data and early 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 this combination therapy.

CircSMARCA5 Is an Upstream Regulator of the Expression of miR-126-3p, miR-515-5p, and Their mRNA Targets, Insulin-like Growth Factor Binding Protein 2 (IGFBP2) and NRAS Proto-Oncogene, GTPase (NRAS) in Glioblastoma.

The involvement of non-coding RNAs (ncRNAs) in glioblastoma multiforme (GBM) pathogenesis and progression has been ascertained but their cross-talk within GBM cells remains elusive. We previously demonstrated the role of circSMARCA5 as a tumor suppressor (TS) in GBM. In this paper, we explore the involvement of circSMARCA5 in the control of microRNA (miRNA) expression in GBM. By using TaqMan® low-density arrays, the expression of 748 miRNAs was assayed in U87MG overexpressing circSMARCA5. Differentially expressed (DE) miRNAs were validated through single TaqMan® assays in: (i) U87MG overexpressing circSMARCA5; (ii) four additional GBM cell lines (A172; CAS-1; SNB-19; U251MG); (iii) thirty-eight GBM biopsies; (iv) twenty biopsies of unaffected brain parenchyma (UC). Validated targets of DE miRNAs were selected from the databases TarBase and miRTarbase, and the literature; their expression was inferred from the GBM TCGA dataset. Expression was assayed in U87MG overexpressing circSMARCA5, GBM cell lines, and biopsies through real-time PCR. TS miRNAs 126-3p and 515-5p were upregulated following circSMARCA5 overexpression in U87MG and their expression was positively correlated with that of circSMARCA5 (r-values = 0.49 and 0.50, p-values = 9 × 10-5 and 7 × 10-5, respectively) in GBM biopsies. Among targets, IGFBP2 (target of miR-126-3p) and NRAS (target of miR-515-5p) mRNAs were positively correlated (r-value = 0.46, p-value = 0.00027), while their expression was negatively correlated with that of circSMARCA5 (r-values = -0.58 and -0.30, p-values = 0 and 0.019, respectively), miR-126-3p (r-value = -0.36, p-value = 0.0066), and miR-515-5p (r-value = -0.34, p-value = 0.010), respectively. Our data identified a new GBM subnetwork controlled by circSMARCA5, which regulates downstream miRNAs 126-3p and 515-5p, and their mRNA targets IGFBP2 and NRAS.

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.

Anticancer effects of ABTL0812, a clinical stage drug inducer of autophagy-mediated cancer cell death, in glioblastoma models.

Glioblastoma multiforme (GBM) is the most malignant adult brain tumor. Current standard of care treatments have very limited efficacy, being the patients´ overall survival 14 months and the 2-year survival rate less than 10%. Therefore, the treatment of GBM is an urgent unmet clinical need. The aim of this study was to investigate in vitro and in vivo the potential of ABTL0812, an oral anticancer compound currently in phase II clinical stage, as a novel therapy for GBM. We showed that ABTL0812 inhibits cell proliferation in a wide panel of GBM cell lines and patient-derived glioblastoma stem cells (GSCs) with half maximal inhibitory concentrations (IC50s) ranging from 15.2 µM to 46.9 µM. Additionally, ABTL0812 decreased GSCs neurosphere formation. GBM cells aggressiveness is associated with a trans-differentiation process towards a less differentiated phenotype known as proneural to mesenchymal transition (PMT). ABTL0812 was shown to revert PMT and induce cell differentiation to a less malignant phenotype in GBM cell lines and GSCs, and consequently reduced cell invasion. As previously shown in other cancer types, we demonstrated that the molecular mechanism of action of ABTL0812 in glioblastoma involves the inhibition of Akt/mTORC1 axis by overexpression of TRIB3, and the activation of endoplasmic reticulum (ER) stress/unfolded protein response (UPR). Both actions converge to induce autophagy-mediated cell death. ABTL0812 anticancer efficacy was studied in vivo using subcutaneous and orthotopic intra-brain xenograft tumor models. We demonstrated that ABTL0812 impairs tumor growth and increases disease-free survival and overall survival of mice. Furthermore, the histological analysis of tumors indicated that ABTL0812 decreases angiogenesis. Finally, we investigated the combination of ABTL0812 with the standard of care treatments for GBM radiotherapy and temozolomide in an orthotopic model, detecting that ABTL0812 potentiates the efficacy of both treatments and that the strongest effect is obtained with the triple combination of ABTL0812+radiotherapy+temozolomide. Overall, the present study demonstrated the anticancer efficacy of ABTL0812 as single agent and in combination with the GBM standard of care treatments in models of glioblastoma and supports the clinical investigation of ABTL0812 as a potential novel therapy for this aggressive brain tumor type.