Glioma Spheres Research Articles

IMMU-17. ADENOSINE DEAMINASE ACTING ON RNA (ADAR) SILENCES TUMOR-INTRINSIC INTERFERON RESPONSE IN DIFFUSE MIDLINE GLIOMA

Abstract INTRODUCTION Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children and is currently incurable. Clinical trials using immune checkpoint blockade (ICB) therapy have failed to show efficacy due to multifactorial mechanisms including limited lymphocyte infiltration, low immune checkpoint (IC) expression, and a low mutational burden. Adenosine deaminase acting on RNA (ADAR) is an RNA editor, which limits anti-tumor immunity by editing/preventing the sensing of endogenous double-stranded RNA (dsRNA). We have found that ADAR protein levels are significantly elevated in DMG patients, suggesting that ADAR’s immune-silencing function is relevant to DMG immune suppression. The onco-histone driver H3K27M promotes epigenetic dysregulation in DMGs, de-suppressing retroelement transcription, a major source of endogenous dsRNA. By inhibiting ADAR, we expect that the increased levels of dsRNA will stimulate intracellular type-1 interferon (IFN) responses, induce chemokine secretion, and promote antitumor CD8+ cytotoxicity. METHODS Human DMG cells and patient-derived glioma spheres were treated with silencing RNA to transiently knockdown (KD) ADAR levels for assays involving in vitro viability and proliferation. Affected pathways were identified using immunoblotting and quantitative PCR. RESULTS Our data shows that ADAR protein levels are elevated in DMG patient proteomic datasets and cell lines. ADAR-depleted patient-derived DMG cell lines exhibit significantly less proliferation compared to control siRNA and ADAR-depleted hTERT-astrocytes (Mann-Whitney test, P-value <0.0001, ****). CONCLUSION Our results demonstrate that DMG tumor cells have an ADAR selective dependency in vitro. In ongoing studies, we aim to determine if ADAR-depletion improves in-vivo survival in both immunodeficient and immunocompetent models in combination with immunotherapies against DMG.

DMRTA2 supports glioma stem-cell mediated neovascularization in glioblastoma

Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Due to its fast proliferation, diffusive growth and therapy resistance survival times are less than two years for patients with IDH-wildtype GBM. GBM is noted for the considerable cellular heterogeneity, high stemness indices and abundance of the glioma stem-like cells known to support tumor progression, therapeutic resistance and recurrence. Doublesex- and mab-3–related transcription factor a2 (DMRTA2) is involved in maintaining neural progenitor cells (NPC) in the cell cycle and its overexpression suppresses NPC differentiation. Despite the reports showing that primary GBM originates from transformed neural stem/progenitors cells, the role of DMRTA2 in gliomagenesis has not been elucidated so far. Here we show the upregulation of DMRTA2 expression in malignant gliomas. Immunohistochemical staining showed the protein concentrated in small cells with high proliferative potential and cells localized around blood vessels, where it colocalizes with pericyte-specific markers. Knock-down of DMRTA2 in human glioma cells impairs proliferation but not viability of the cells, and affects the formation of the tumor spheres, as evidenced by strong decrease in the number and size of spheres in in vitro cultures. Moreover, the knockdown of DMRTA2 in glioma spheres affects the stabilization of the glioma stem-like cell-dependent tube formation in an in vitro angiogenesis assay. We conclude that DMRTA2 is a new player in gliomagenesis and tumor neovascularization and due to its high expression in malignant gliomas could be a biomarker and potential target for new therapeutic strategies in glioblastoma.

IMMU-30. NOVEL HYPOMETHYLATING AGENT GSK-3484862 SENSITIZES GLIOBLASTOMA TO NY-ESO-1 SPECIFIC IMMUNOTHERAPY

Abstract INTROUCTION Immunotherapy is a promising mode of treatment due to its specificity and efficacy in eliminating tumors, but is limited in GBM due to the lack of immunogenicity and unique cancer antigen expression. Previous work from our group has shown that Decitabine (DAC), a demethylating agent, can improve the immunogenicity of Glioblastoma (GBM) for immuno-therapeutic targeting through the upregulation of immunogenic cancer testis antigens (CTA) such as NY-ESO-1 and altered expression of other immunoregulatory programs. However, immunosensitization using DAC is limited due to non-specific toxicity after prolonged administration. We hypothesize that a newly synthesized hypomethylating agent, GSK-3484862 (GSK), may be an effective alternative to DAC in promoting CTA expression in GBM with limited cytotoxic effects on surrounding immune cells. METHODS Human GBM cells and patient-derived glioma spheres were treated with GSK and DAC. We investigated the differential expression in NY-ESO-1 at a transcriptional level between DAC and GSK through quantitative real-time PCR. To assess whether these differences translated into heightened immune responses as a result of NY-ESO-1 upregulation, we co-cultured DAC and GSK-treated immortalized and primary cell lines with transduced NY-ESO-1 specific T-cell receptor T-cells. RESULTS Immortalized and primary glioma sphere cells demonstrated equivalent NY-ESO-1 gene upregulation after four days of treatment between 1 µM DAC and 5 µM GSK. Glioma spheres treated for three weeks at a dose of either 0.5 µM DAC or 2.5 µM GSK demonstrate equivalent NY-ESO-1 upregulation. Furthermore, xCELLigence data demonstrates that equivalent NY-ESO-1 expression achieved by the differential dosing between GSK and DAC leads to similar rates of NY-ESO-1 specific T-cell mediated killing, indicating a functional equivalence between GSK and DAC induced immunosensitization at equivalent NY-ESO-1 expression levels. CONCLUSION Our results demonstrate a preclinical rationale for the use of GSK as a viable pro-antigenic agent for the sensitization of GBM to targeted immune therapy.

Thermo-Chemical Resistance to Combination Therapy of Glioma Depends on Cellular Energy Level.

Thermal therapy has been widely used in clinical tumor treatment and more recently in combination with chemotherapy, where the key challenge is the treatment resistance. The mechanism at the cellular level underlying the resistance to thermo-chemical combination therapy remains elusive. In this study, we constructed 3D culture models for glioma cells (i.e., 3D glioma spheres) as the model system to recapitulate the native tumor microenvironment and systematically investigated the thermal response of 3D glioma spheres at different hyperthermic temperatures. We found that 3D glioma spheres show high viability under hyperthermia, especially under high hyperthermic temperatures (42 °C). Further study revealed that the main mechanism lies in the high energy level of cells in 3D glioma spheres under hyperthermia, which enables the cells to respond promptly to thermal stimulation and maintain cellular viability by upregulating the chaperon protein Hsp70 and the anti-apoptotic pathway AKT. Besides, we also demonstrated that 3D glioma spheres show strong drug resistance to the thermo-chemical combination therapy. This study provides a new perspective on understanding the thermal response of combination therapy for tumor treatment.

MAPK9 is Correlated with a Poor Prognosis and Tumor Progression in Glioma

Glioma has a high incidence in young and middle-aged adults and a poor prognosis. Because of late diagnosis and uncontrollable recurrence of the primary tumor after failure of existing treatments, glioma patients tend to have a poor prognosis. Recent advances in research have revealed that gliomas exhibit unique genetic features. Mitogen-activated protein kinase 9 (MAPK9) is significantly upregulated in mesenchymal glioma spheres and may be a new target for glioma diagnosis. This study aimed to investigate the potential diagnostic significance and predictive value of MAPK9 in glioma. Paraffin-embedded tumor tissues and paracancerous tissues were collected from 150 glioma patients seen at the General Hospital of Northern Theater Command. Immunohistochemistry and western blot assays were used to detect the expression levels of MAPK9. Prognosis and survival analyses were performed using SPSS 26 software for univariate/multivariate analysis and log-rank analysis. Cellular models were used to assess the effect of MAPK9 overexpression and knockdown in vitro. MAPK9 expression was higher in glioma tissues than in paraneoplastic tissues. Prognostic and survival analyses revealed that the MAPK9 expression level is an independent prognostic factor in glioma patients. In addition, overexpression of MAPK9 significantly promoted the proliferation and migration of primary glioma cells, possibly via the Wnt/β-catenin-regulated EMT pathway. MAPK9 is an independent prognostic factor in glioma and is involved in tumor progression.

Abstract 3459: Patient derived gliomaspheres exhibit HA concentration dependent invasiveness in 3D hydrogels

Abstract Hyaluronic acid (HA) is a highly abundant glycosaminoglycan within the central nervous system. In glioblastoma (GBM), interactions with HA mediate tumor cell invasion. An improved knowledge of the biological underpinnings of HA-dependent GBM cell migration can facilitate the development of efficacious therapeutics. To better study how HA in the tumor matrix affects invasion, we have developed a tunable, 3D culture system in which to study gliomaspheres (GSs) of patient-derived tumor cells. Photopolymerization (365 nm for 15 seconds) of GSs suspended in a solution 0.025 w/v% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), thiolated 4-arm poly-ethylene glycol (20 kDa, Laysan Bio), 8-arm poly-ethylene glycol-norbornene (20 kDa, Laysan Bio), thiolated peptides containing an ‘RGD’ amino acid motif, and either 0.10, 0.25, 0.50, or 0.75 w/v% of thiolated HA (700 kDa average molecular weight, Lifecore Biomedical) yielded 3D hydrogels of varying HA concentrations and similar mechanical properties. Relative invasive capacity, quantified by shape factor (deviation from circularity) and migration length (maximum extension length of processes from the GS periphery), differed across patient lines and was independent of their Cancer Genome Atlas (TCGA) classification as either mesenchymal or proneural. Hydrogels with higher amounts of HA (>0.25% w/v) generally enhanced invasion as compared to low HA (0.10 w/v%) hydrogels. In certain cell lines, a biphasic relationship between HA concentration and migration was observed, in line with previous reports of CD44 expression and migration. Moreover, inhibition of the CD44-Ezrin-Actin axis, using NSC668394, reduced migratory activity in hydrogels with higher HA (>0.25% w/v), while slightly increasing invasion in hydrogels with low HA (0.10 w/v%). In sum, results demonstrate the use of a matrix-mimetic, 3D hydrogel system in which to study GBM invasion through the local extracellular matrix. Citation Format: Gevick Safarians, Itay Solomon, Alireza Sohrabi, Stephanie Seidlits. Patient derived gliomaspheres exhibit HA concentration dependent invasiveness in 3D hydrogels [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 3459.

CTNI-07. ABTC-1701: PILOT SURGICAL PK STUDY OF BGB324 (BEMCENTINIB) IN RECURRENT GLIOBLASTOMA PATIENTS – RESULTS FROM INTERIM FUTILITY ANALYSIS

Abstract BACKGROUND Glioblastoma often can relapse as mesenchymal (MES) tumors, indicating a phenotypic shift during clinical progression. Glioma spheres, when they gain MES phenotypes, develop dependence on AXL for their growth both in vitro and in vivo. The first-in-class orally bioavailable AXL kinase inhibitor bemcentinib has IC50 of 14 nM and is > 100-fold selective for AXL over other kinases. Bemcentinib is currently under evaluation as a monotherapy and in combination with other treatments across various PhII trials in several oncology indications. METHODS This is an open-label, multicenter, intratumoral pharmacokinetic study of bemcentinib in patients with recurrent glioblastoma for whom a surgical resection is medically indicated. All subjects must have had histological confirmation of glioblastoma by either biopsy or resection that is progressive or recurrent following radiation therapy ± chemotherapy. Intratumoral drug levels were analyzed by LC/MS. RESULTS A planned analysis after the first 5 patients were enrolled with glioblastoma (4 IDH WT and 1 IDH-mutant), (3m, 2f, median age 57, KPS 80). Bemcentinib concentration in contrast enhancing brain tissue ranged from 3.1 to 43.0 uM with a geometric mean concentration of 11.1 uM (% CV, 132.1). The drug concentration in contrast enhancing tumor tissue exceeded the 1.0 uM threshold in all 5 patients, satisfying the criteria of the protocol to enroll an additional 15 patients into the surgical study. Total drug levels were approximately 2-fold greater in contrast enhancing tissue as compared to tissue from a non-enhancing region of the tumor (geometric mean, 5.8 uM; % CV, 187.7). The mean ratio of the drug concentration in brain tissue to plasma was 25.9 (% CV, 92.7) for contrast enhancing tissue and 13.4 (% CV, 126.8) for non-enhancing tissue. CONCLUSIONS Bemcentinib readily distributes in brain tumor tissue following oral administration. The study continues to complete accrual for the remaining 15 patients.

Effects of the DRD2/3 antagonist ONC201 and radiation in glioblastoma

BackgroundGlioblastoma (GBM) is the deadliest of all brain cancers in adults. The current standard-of-care is surgery followed by radiotherapy and temozolomide, leading to a median survival time of only 15 months. GBM are organized hierarchically with a small number of glioma-initiating cells (GICs), responsible for therapy resistance and tumor recurrence, suggesting that targeting GICs could improve treatment response. ONC201 is a first-in-class anti-tumor agent with clinical efficacy in some forms of high-grade gliomas. Here we test its efficacy against GBM in combination with radiation. MethodsUsing patient-derived GBM lines and mouse models of GBM we test the effects of radiation and ONC201 on GBM self-renewalin vitro and survivalin vivo.A possible resistance mechanism is investigated using RNA-Sequencing. ResultsTreatment of GBM cells with ONC201 reduced self-renewal, clonogenicity and cell viabilityin vitro. ONC201 exhibited anti-tumor effects on radioresistant GBM cells indicated by reduced self-renewal in secondary and tertiary glioma spheres. Combined treatment of ONC201 and radiation prolonged survival in syngeneic and patient-derived orthotopic xenograft mouse models of GBM. Subsequent transcriptome analyses after combined treatment revealed shifts in gene expression signatures related to quiescent GBM populations, GBM plasticity, and GBM stem cells. ConclusionsOur findings suggest that combined treatment with the DRD2/3 antagonist ONC201 and radiation improves the efficacy of radiation against GBMin vitroandin vivothrough suppression of GICs without increasing toxicity in mouse models of GBM. A clinical assessment of this novel combination therapy against GBM is further warranted.

Tumor Microenvironment Is Critical for the Maintenance of Cellular States Found in Primary Glioblastomas.

Glioblastoma (GBM), an incurable tumor, remains difficult to model and more importantly to treat due to its genetic/epigenetic heterogeneity and plasticity across cellular states. The ability of current tumor models to recapitulate the cellular states found in primary tumors remains unexplored. To address this issue, we compared single-cell RNA sequencing of tumor cells from 5 patients across four patient-specific glioblastoma stem cell (GSC)-derived model types, including glioma spheres, tumor organoids, glioblastoma cerebral organoids (GLICO), and patient-derived xenografts. We find that GSCs within the GLICO model are enriched for a neural progenitor-like cell subpopulation and recapitulate the cellular states and their plasticity found in the corresponding primary parental tumors. These data demonstrate how the contribution of a neuroanatomically accurate human microenvironment is critical and sufficient for recapitulating the cellular states found in human primary GBMs, a principle that may likely apply to other tumor models. SIGNIFICANCE: It has been unclear how well different patient-derived GBM models are able to recreate the full heterogeneity of primary tumors. Here, we provide a complete transcriptomic characterization of the major model types. We show that the microenvironment is crucial for recapitulating GSC cellular states, highlighting the importance of tumor-host cell interactions.See related commentary by Luo and Weiss, p. 907.This article is highlighted in the In This Issue feature, p. 890.

Effects of progesterone on the cell number of gliomaspheres derived from human glioblastoma cell lines

AimsThe malignancy of the Glioblastomas (GBM), the most frequent and aggressive brain tumors, have been associated with the presence of glioma stem cells (GSCs) which can form gliomaspheres (GS) in vitro. Progesterone (P) increases the proliferation, migration, and invasion of GBM cell lines through the interaction with its intracellular receptor (PR). However, it is unknown if the PR is expressed and the possible effects of P in the formation/differentiation of GS. Main methodsGS were grown from U251 and U87 cell lines by selective culture with serum-free neural stem cell medium. GSCs were identified by the detection of Sox2, Ki67, Nestin, CD133, and CD15 by immunofluorescence. Additionally, the relative expression of PROM1, NES, SOX2, OLIG2, EZH2, BMI1 and PR genes was evaluated by RT-qPCR. The GS were treated with P, and the number of cells was quantified. By RT-PCR the βIII-TUB and GFAP differentiation genes were evaluated. Key findingsGS were maintained until passage four. The expression of all GSCs markers was significantly higher in GS as compared with the basal culture of U251 and U87 cells. We demonstrated for the first time that PR is expressed in GS and this expression was higher as compared with the U251 and U87 cells in basal conditions. Also, we observed that P increased the number of cells derived primary gliomaspheres (GS1) from the U251 line, as well as the expression of the neuronal differentiation marker βIII-TUB. SignificanceThese results suggest the participation of P in the growth of GSCs.

Overexpression of HGF/MET axis along with p53 inhibition induces de novo glioma formation in mice.

BackgroundAberrant MET receptor tyrosine kinase (RTK) activation leads to invasive tumor growth in different types of cancer. Overexpression of MET and its ligand hepatocyte growth factor (HGF) occurs more frequently in glioblastoma (GBM) than in low-grade gliomas. Although we have shown previously that HGF-autocrine activation predicts sensitivity to MET tyrosine kinase inhibitors (TKIs) in GBM, whether it initiates tumorigenesis remains elusive.MethodsUsing a well-established Sleeping Beauty (SB) transposon strategy, we injected human HGF and MET cDNA together with a short hairpin siRNA against Trp53 (SB-hHgf.Met.ShP53) into the lateral ventricle of neonatal mice to induce spontaneous glioma initiation and characterized the tumors with H&E and immunohistochemistry analysis. Glioma sphere cells also were isolated for measuring the sensitivity to specific MET TKIs.ResultsMixed injection of SB-hHgf.Met.ShP53 plasmids induced de novo glioma formation with invasive tumor growth accompanied by HGF and MET overexpression. While glioma stem cells (GSCs) are considered as the tumor-initiating cells in GBM, both SB-hHgf.Met.ShP53 tumor sections and glioma spheres harvested from these tumors expressed GSC markers nestin, GFAP, and Sox 2. Moreover, specific MET TKIs significantly inhibited tumor spheres’ proliferation and MET/MAPK/AKT signaling.ConclusionsOverexpression of the HGF/MET axis along with p53 attenuation may transform neural stem cells into GSCs, resulting in GBM formation in mice. These tumors are primarily driven by the MET RTK pathway activation and are sensitive to MET TKIs. The SB-hHgf.Met.ShP53 spontaneous mouse glioma model provides a useful tool for studying GBM tumor biology and MET-targeting therapeutics.

Irradiation enhances the therapeutic effect of the oncolytic adenovirus XVir-N-31 in brain tumor initiating cells.

Virotherapy using oncolytic viruses is an upcoming therapy strategy for cancer treatment. A variety of preclinical and clinical trials have indicated that adenoviruses may be used as potent agents in the treatment of a variety of cancers, and also for the treatment of brain tumors. In these studies, it has also been shown that oncovirotherapy is safe in terms of toxicity and side effects. In addition, previous studies have presented evidence for a significant role of oncovirotherapy in the activation of anti-tumor immune responses. With regard to oncolytic adenoviruses, we have demonstrated previously that the multifunctional protein Y-box binding protein-1 (YB-1) is a potent factor that was used to develop an YB-1-dependent oncolytic adenovirus (XVir-N-31). XVir-N-31 provides the opportunity for tumor-selective replication and exhibited marked oncolytic properties in a mouse glioma tumor model using therapy-resistant brain tumor initiating cells (BTICs). In a number of, but not all, patients with glioma, YB-1 is primarily located in the nucleus; this promotes XVir-N-31-replication and subsequently tumor cell lysis. However, in certain BTICs, only a small amount of YB-1 has been identified to be nuclear, and therefore virus replication is suboptimal. YB-1 in BTICs was demonstrated to be translocated into the nucleus following irradiation, which was accompanied by an enhancement in XVir-N-31 production. R28 glioma spheres implanted in living organotypic human brain slices exhibited a significantly delayed growth rate when pre-irradiated prior to XVir-N-31-infection as compared with single treatment methods. Consistent with the in vitro data, R28 glioma-bearing mice exhibited a prolonged mean and median survival following single tumor irradiation prior to intratumoral XVir-N-31 injection, compared with the single treatment methods. In conclusion, the present study demonstrated that in an experimental glioma model, tumor irradiation strengthened the effect of an XVir-N-31-based oncovirotherapy.

Isolation and characterization of exosomes from IDH mutant gliomas.

152 Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are found in > 70% of intermediate grade gliomas. Gain-of-function mutations in IDH1 promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and in body fluids of patients. TEX serve as a communication system between tumor and normal cells. Circulating TEX carry a complex molecular and genetic cargo and interfere with functions of immune cells. This study characterizes TEX produced by IDH mutant glioma spheres and TEX from plasma of patients with IDH mutant gliomas. Methods: TEX produced by patient-derived IDH1(mut) or IDH1(wt) glioma spheres or TEX in patient’s plasma were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers and molecular profile were characterized and the crossing of the blood-brain barrier (BBB) and biodistribution was investigated by in vivo imaging of TEX. Interactions of TEX with endothelial cells, astrocytes and immune cells were demonstrated by confocal microscopy. Results: TEX isolated from supernatants of IDH1(mut) or IDH1(wt) glioma spheres by mini-SEC carried TSG101 and showed the typical size and vesicular morphology of exosomes. BCA protein assays and qNano analysis revealed an elevated exosome production by IDH mutant cells ( p< 0.01). TEX carried immunosuppressive proteins (FasL, CTLA-4 and TRAIL) and IDH mutant exosomes carried higher levels of these proteins and of adenosine pathway components CD39 and CD73. Labeled TEX injected in the white matter of mice crossed the BBB and were detectable in distant organs 24h after injection. TEX were rapidly internalized by endothelial cells and astrocytes, but not by T cells, which interacted with TEX by surface signaling. These results were confirmed by investigating TEX isolated from glioma patient’s plasma. Conclusions: The data suggest that TEX play an important role in IDH mutant gliomas and drive tumor progression in part by inducing suppression of immune cells. Future efforts will focus on characterizing immunosuppressive effects of TEX derived from IDH mutant cells vs gliomas without IDH mutations.

Proteins of the Wnt signaling pathway as targets for the regulation of CD133+ cancer stem cells in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most aggressive types of brain tumor and is highly resistant to therapy. The median survival time for patients with GBM is 15 months. GBM resistance to treatment is associated with cancer stem cells (CSCs). CD133 membrane glycoprotein is the best‑known marker of GBM CSCs. The Wnt signaling pathway plays an important role in the proliferation of all stem cells. To the best of our knowledge, the present study was the first to examine the expression levels of proteins associated with the Wnt signaling pathway in СD133+ CSCs of human GBM. Furthermore, potential targets that may regulate СD133+ CSCs in human GBM were investigated. The human GBM U‑87MG cell line was cultured in neurobasal medium supplemented with B27, fibroblast growth factor, epidermal growth factor and no serum. Immunohistochemical characteristics of glioma spheres were investigated based on the expression of key markers of CSCs. CD133+ cells were extracted from glioma spheres by cell sorting and then lysed. High‑performance liquid chromatography‑mass spectrometry was used for proteome analysis. Lysates of CD133‑ cells in GBM were used for comparison. The present study was the first to describe the conceptual proteome differences between GBM and CD133+ CSCs of the common pool. Major differences were identified in the glycolysis/gluconeogenesis, focal adhesion, tight junction and Wnt signaling pathways. This study aimed to analyze the crucial role that proteins of the Wnt signaling pathway play in stem cell proliferation. The identified proteins were analyzed for their association with the Wnt signaling pathway using the international open databases PubMed, Protein Analysis Through Evolutionary Relationships, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and Search Tool for the Retrieval of Interacting Genes/Proteins. An increased expression of 12 proteins associated with the Wnt signaling pathway were identified in GBM CD133+ CSCs, which included catenin β‑1, disheveled associated activator of morphogenesis 1, RAC family small GTPase 2 and RAS homolog gene family member A, a number of which are also associated with adherens junctions. The Wnt signaling pathway is not upregulated in CSCs; however, the high expression levels of adenomatous polyposis coli, β‑catenin, C‑terminal binding protein (CtBP) and RuvB‑like AAA ATPase 1 (RUVBL1 or Pontin52) proteins suggest the possibility of alternative activation of specific genes in the nuclei of these cells. Calcyclin‑binding protein, casein kinase II α, casein kinase II β, CtBP1, CtBP2, CUL1 and RUVBL1 proteins may be used as targets for the pharmaceutical regulation of CSCs in complex GBM treatment.

Exploitation of 2-Hydroxyglutarate-Mediated Deficiency in Homologous Recombination with PARP Inhibition to Achieve Radiosensitization in IDH-Mutant Tumors

Recent work has demonstrated deficient HR in IDH1/2-mutant (mt) cells, conferring susceptibility to synthetic lethality with PARP inhibitors (PARPi). We conducted translational studies to assess whether radiation (RT) + PARPi is selectively more potent in IDH-mt glioma and cholangiocarcinoma cells, both of which commonly harbor IDH mutations in humans. In vitro/in vivo treatment with concurrent RT + PARPi was tested in IDH-mt and -wild-type (wt) settings compared to RT alone, PARPi alone, and vehicle control (VEH). Paired cell lines included: (1) IDH1-mt TS603 and IDH1-wt TS543 human glioma spheres; (2) isogenic IDH1-mt and -wt immortalized human astrocytes (IHAs). Two in vivo models were used: (1) nude mouse orthotopic xenografts where 5 x 105 TS603 or TS543 cells were stereotactically implanted in the right striatum of the brain; (2) a de novo PDGF-driven murine glioma model using the RCAS/tva system [ref 3] to generate isogenic IDH1-mt or -wt gliomas in Ntva mice, which express the tva receptor from the nestin promoter, allowing gene transfer to CNS progenitors. Treatment started in nude mice once tumors reached threshold BLI signal of ROI = 2 x 105 units and in Ntva mice at 5 wks post stereotactic coinjection of DF1 cells producing RCAS-PDGF and either RCAS-IDH1-wt-shp53 or RCAS-IDH1-mt-shp53 into the right striatum. RT was 2 Gy x 5 fractions. Veliparib (25 mg/kg IP) and VEH were given daily until demise. Viability and clonogenic survival were decreased with RT + PARPi vs RT and with PARPi vs VEH among TS603 cells and IDH1-mt IHAs (all p<0.05), but not TS543 cells and IDH1-wt IHAs (all p>0.05). Increased DNA damage was observed on neutral comet assay in IDH1-mt vs -wt IHAs after treatment with RT + PARPi vs RT and after PARPi vs VEH (all p<0.05). An IDH1-mt-specific survival benefit of RT + PARPi vs RT was observed in both mouse models (nude: median [mdn] 20 vs 10 d, p=0.002; Ntva: mdn 70 vs 18 d, p=0.006), and of PARPi vs VEH in Ntva mice (mdn 15 vs 10 d, p=0.05) with a trend towards longer survival in nude mice bearing IDH1-mt tumors (mdn 9 vs 7 d, p=0.14). No survival benefit was observed for RT + PARPi vs RT nor PARPi vs VEH in the IDH1-wt setting (all p>0.05). CR was observed only among nude mice bearing IDH1-mt tumors after RT + PARPi (n=4/13). Experiments for paired human IDH-mt and -wt cholangiocarcinoma cell lines are ongoing. γ-H2AX staining of resection specimens revealed more foci at baseline in IDH-mt flash frozen gliomas (n=6) and FFPE cholangiocarcinomas (n=3) vs IDH-wt specimens (n=6, p=0.002 and n=3, p=0.005, respectively), confirming applicability of this approach in human tumors. RT + PARPi demonstrates increased efficacy selectively against IDH1-mt gliomas, suggesting that clinical trials are warranted and that this approach may have relevance in the IDH-mt setting across histologies.

SOX9-PDK1 axis is essential for glioma stem cell self-renewal and temozolomide resistance.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor with limited therapeutic options. Glioma stem cell (GSC) is thought to be greatly responsible for glioma tumor progression and drug resistance. But the molecular mechanisms of GSC deriving recurrence and drug resistance are still unclear. SOX9 (sex-determining region Y (SRY)-box9 protein), a transcription factor expressed in most solid tumors, is reported as a key regulator involved in maintaining cancer hallmarks including the GSCs state. Previously, we have observed that silencing of SOX9 suppressed glioma cells proliferation both in vitro and in vivo. Here, we found that SOX9 was essential for GSC self-renewal. Silencing of SOX9 down-regulated a broad range of stem cell markers and inhibited glioma cell colony and sphere formation. We identified pyruvate dehydrogenase kinase 1 (PDK1) as a target gene of SOX9 using microarray analyses. PDK1 inactivation greatly inhibited glioma cell colony and sphere formation and sensitized glioma spheres to temozolomide (TMZ) toxicity. In addition, SOX9-shRNA and PDK1 inhibitor could greatly sensitize GSC to TMZ in vivo. Taken together, our data reveals that SOX9-PDK1 axis is a key regulator of GSC self-renewal and GSC temozolomide resistance. These findings may provide help for future human GBM therapy.

Stanniocalcin-1 augments stem-like traits of glioblastoma cells through binding and activating NOTCH1

Glioblastoma (GBM) is a fatal tumor and comprises heterogeneous cells in which a subpopulation with stem cell-like properties is included. Cancer cells with stem cell-like properties account for tumor initiation, drug resistance and recurrence. To identify and characterize specific factors in regulating stem-like traits is critical for GBM therapeutic. Here, we showed that Stanniocalcin-1 (STC1), a secretory glycoprotein, functions as a novel stimulator for stem-like traits of GBM cells. We found STC1 was prominently expressed in glioma spheres which are mainly comprised of glioma stem-like cells. The stem-like traits of GBM cells, as determined by the expression of stem cell markers, tumor-sphere formation efficiency and colony-forming ability, were enhanced by STC1 overexpression and inhibited by STC1 knockdown. Furthermore, introduction of STC1 enhanced tumorigenesis in vivo while knockdown of STC1 showed reverse effect. Finally, we demonstrated that STC1 interacted with the extracellular domain of NOTCH1 to activate NOTCH1-SOX2 signaling pathway, by which STC1 augmented the stem-like traits of GBM cells. Taken together, our data herein indicate that STC1 is a novel non-canonical NOTCH ligand and acts as a crucial regulator of stemness in GBM.

Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2.

Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity-dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.

Temozolomide increases MHC-I expression via NF-κB signaling in glioma stem cells.

Gliomas are the most common and primary tumors of the central nervous system in adults. Temozolomide (TMZ) is the main drug used to treat glioma; however, prognosis remains poor for most patients. Glioma stem cells (GSCs) are thought to enable glioma initiation and evasion from immune surveillance; their immunogenicity can be determined by expression of major histocompatibility complex (MHC)-I. The present study investigated the effect of TMZ on MHC-I expression in GSCs. Glioma spheres were cultured in serum-free medium containing epidermal growth factor, basic fibroblast growth factor, and B27; MHC-I expression was detected by immunocytochemistry, quantitative real-time PCR, and flow cytometry. Nuclear factor (NF)-κB expression in glioma stem cells was detected by Western blot. TMZ enhanced MHC-I expression in GSCs, and NF-κB was activated. TMZ treatment increased MHC-I expression via modulation of NF-κB signaling in GSCs. In addition to being a chemotherapeutic agent, TMZ may also serve as an immunomodulatory agent in the treatment of glioma patients.

The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma.

Osteopontin (SPP1, a secreted phosphoprotein 1) is primarily involved in immune responses, tissue remodelling and biomineralization. However, it is also overexpressed in many cancers and regulates tumour progression by increasing migration, invasion and cancer stem cell self-renewal. Mechanisms of SPP1 overexpression in gliomas are poorly understood. We demonstrate overexpression of two out of five SPP1 isoforms in glioblastoma (GBM) and differential isoform expression in glioma cell lines. Up-regulated SPP1 expression is associated with binding of the GLI1 transcription factor to the promoter and OCT4 (octamer-binding transcription factor 4) to the first SPP1 intron of the SPP1 gene in human glioma cells but not in non-transformed astrocytes. GLI1 knockdown reduced SPP1 mRNA and protein levels in glioma cells. GLI1 and OCT4 are known regulators of stem cell pluripotency. GBMs contain rare cells that express stem cell markers and display ability to self-renew. We reveal that SPP1 is overexpressed in glioma initiating cells defined by high rhodamine 123 efflux, sphere forming capacity and stemness marker expression. Forced differentiation of human glioma spheres reduced SPP1 expression. Knockdown of SPP1, GLI1 or CD44 with siRNAs diminished sphere formation. C6 glioma cells stably depleted of Spp1 displayed reduced sphere forming capacity and downregulated stemness marker expression. Overexpression of the wild type Spp1, but not Spp1 lacking a Cd44 binding domain, rescued cell ability to form spheres. Our findings show re-activation of the embryonic-type transcriptional regulation of SPP1 in malignant gliomas and point to the importance of SPP1-CD44 interactions in self-renewal and pluripotency glioma initiating cells.