Meningioma Cell Lines Research Articles

Programmed death ligand-1 (PD-L1) expression in meningioma; prognostic significance and its association with hypoxia and NFKB2 expression

Management of clinically aggressive meningiomas is a considerable challenge. PD-L1 induced immune suppression has increasingly gained attention in clinical management of cancer; however, to date, the clinical significance and regulatory mechanisms of PD-L1 in meningioma is not yet fully characterized. We sought to characterize PD-L1 expression in meningioma and elucidate its regulatory mechanisms. Immunohistochemical staining of PD-L1 expression in meningiomas showed 43% positivity in both tumor and immune cells and we observed intra and inter tumoral heterogeneity. Univariate and multivariate analyses confirmed that PD-L1 protein expression is an independent prognostic marker for worse recurrence free survival in meningioma. Furthermore, our transcriptomic analysis revealed a strong association between PD-L1 expression and that of NFKB2 and carbonic anhydrase 9 (CA9). We also demonstrated that both of these markers, when co-expressed with PD-L1, predict tumor progression. Our studies on several meningioma cell lines cultured in hypoxic conditions validated the association of CA9 and PD-L1 expression. Here we show the clinical significance of PD-L1 in meningioma as a marker that can predict tumor recurrence. We also show an association PD-L1 expression with NFKB2 expression and its induction under hypoxic conditions. These findings may open new avenues of molecular investigation in pathogenesis of meningioma.

Abstract 6384: TEAD inhibition reduces tumor proliferation in Merlin null meningioma and schwannoma

Abstract Introduction: Meningiomas are the most common intracranial tumor and arise from the arachnoidal cell layer of the meninges. Schwannomas develop from Schwann cells which provide myelin and trophic support in the peripheral nerves. The tumor suppressor Merlin is deleted in approximately 50-60% of meningiomas and 70% of schwannomas. Loss of Merlin results in dysregulation of the Hippo pathway and consequent aberrant activation of the transcriptional coactivators YAP and TAZ. Nuclear localisation of YAP and TAZ has been shown to drive tumor phenotypes in many cancers. The TEAD family of transcription factors are major targets of YAP and TAZ-dependent signaling. TEAD family members undergo autopalmitoylation and this modification is required for transcriptional activation by YAP or TAZ. Vivace therapeutics have identified small molecule inhibitors of autopalmitoylation that block the interaction of TEAD family members with YAP and TAZ. This study seeks to establish the efficacy of small molecule inhibitors of TEAD activity in meningioma and schwannoma tumor cells. Experimental Procedures: Meningioma cell lines, primary human meningioma and schwannoma cells, as well as a schwannoma mouse model (PerisotinCRE /NF2 fl/fl) were treated with Vivace inhibitors to assess their effects upon tumor cell proliferation and TEAD driven transcription. Results: TEAD inhibition significantly reduced the proliferation of the BenMen-1 meningioma cell line, as well as in primary human meningioma cells. In schwannoma, inhibitors reduced proliferation and also reduced the expression of CTGF, a target of TEAD activation in both primary human meningioma and schwannoma cells. These novel inhibitors are also currently being tested in the Periostin CRE-NF2fl/fl schwannoma mouse model to evaluate their efficacy in vivo. Conclusions: The use of these novel compounds to inhibit TEAD function shows great potential as an effective treatment for reducing the rate of cell proliferation in meningioma and schwannoma, two clinically important tumor types. Citation Format: Liyam Laraba, Julio Grimm de Guibert, Tracy T. Tang, Leonard Post, David B. Parkinson. TEAD inhibition reduces tumor proliferation in Merlin null meningioma and schwannoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6384.

Characterization and comparison of genomic profiles between primary cancer cell lines and parent atypical meningioma tumors

BackgroundMeningiomas are the second most common primary tumors of the central nervous system. However, there is a paucity of data on meningioma biology due to the lack of suitable preclinical in vitro and in vivo models. In this study, we report the establishment and characterization of patient-derived, spontaneously immortalized cancer cell lines derived from World Health Organization (WHO) grade I and atypical WHO grade II meningiomas.MethodsWe evaluated high-resolution 3T MRI neuroimaging findings in meningioma patients which were followed by histological analysis. RT-qPCR and immunostaining analyses were performed to determine the expression levels of meningioma-related factors. Additionally, flow cytometry and sorting assays were conducted to investigate and isolate the CD133 and CD44 positive cells from primary atypical meningioma cells. Further, we compared the gene expression profiles of meningiomas and cell lines derived from them by performing whole-exome sequencing of the blood and tumor samples from the patients, and the primary cancer cell lines established from the meningioma tumor.ResultsOur results were consistent with earlier studies that reported mutations in NF2, SMO, and AKT1 genes in atypical meningiomas, and we also observed mutations in MYBL2, a gene that was recently discovered. Significantly, the genomic signature was consistent between the atypical meningioma cancer cell lines and the tumor and blood samples from the patient.ConclusionOur results lead us to conclude that established meningioma cell lines with a genomic signature identical to tumors might be a valuable tool for understanding meningioma tumor biology, and for screening therapeutic agents to treat recurrent meningiomas.

Boron Neutron Capture Therapy and Photodynamic Therapy for High-Grade Meningiomas.

Meningiomas are the most common type of intracranial brain tumors in adults. The majority of meningiomas are benign with a low risk of recurrence after resection. However, meningiomas defined as grades II or III, according to the 2016 World Health Organization (WHO) classification, termed high-grade meningiomas, frequently recur, even after gross total resection with or without adjuvant radiotherapy. Boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) are novel treatment modalities for malignant brain tumors, represented by glioblastomas. Although BNCT is based on a nuclear reaction and PDT uses a photochemical reaction, both of these therapies result in cellular damage to only the tumor cells. The aim of this literature review is to investigate the possibility and efficacy of BNCT and PDT as novel treatment modalities for high-grade meningiomas. The present review was conducted by searching PubMed and Scopus databases. The search was conducted in December 2019. Early clinical studies of BNCT have demonstrated activity for high-grade meningiomas, and a phase II clinical trial is in progress in Japan. As for PDT, studies have investigated the effect of PDT in malignant meningioma cell lines to establish PDT as a treatment for malignant meningiomas. Further laboratory research combined with proper controlled trials investigating the effects of these therapies is warranted.

Chapter 8 - Preclinical models of meningioma: Cell culture and animal systems

Meningioma is the most common primary intracranial tumor; yet there are no effective systemic or molecular therapies for meningioma patients. One of the primary barriers to understanding meningioma biology and identifying novel therapeutic targets is the lack of tractable preclinical models. While numerous model systems have been created for meningioma, many have fundamental drawbacks. This chapter details the strengths and limitations of existing meningioma models and suggests possible future model systems. Cell culture meningioma models consist of human meningioma cell lines derived from tumor resection specimens, but unfortunately, in vitro systems do not capture the histologic architecture, the tumor microenvironment, or the heterogeneity of meningiomas. Mouse meningioma systems range from genetically engineered mouse models (GEMMs) to patient-derived xenografts (PDXs) and overcome some of the limitations of cultured meningioma cells. However, many in vivo systems have poor reproducibility or fail to recapitulate important aspects of meningioma biology, such as tumor latency. Despite these drawbacks, new discoveries in meningioma biology and advances in the technologies used to develop model systems provide hope that more representative models of meningioma will be developed in the near future.

The CREB-binding protein inhibitor ICG-001: a promising therapeutic strategy in sporadic meningioma with NF2 mutations.

BackgroundMeningiomas with Neurofibromin 2 gene mutations (NF2-mutant meningiomas) account for ~40% of the sporadic meningiomas. However, there is still no effective drug treatment for the disease.MethodsExpression profile of Merlin protein was explored through immunohistochemistry in a meningioma patient cohort (n = 346). A 20-agent library covering a wide range of meningioma relevant targets was tested using meningioma cell lines IOMM-Lee (NF2 wildtype) and CH157-MN (NF2 deficient). Therapeutic effects and biological mechanisms of the identified compound, ICG-001, in NF2-mutant meningiomas were further characterized in vitro and in patient-derived xenograft (PDX) models.ResultsLow Merlin expression was associated with meningioma proliferation and poor clinical outcomes in a large patient series. ICG-001, a cAMP-responsive element binding (CREB)-binding protein (CBP) inhibitor, selectively suppressed tumor growth of cells with low Merlin expression. Besides, ICG-001 mediated CH157-MN and IOMM-Lee growth inhibition primarily through robust induction of the G1 cell-cycle arrest. Treatment with ICG-001 alone significantly reduced the growth of NF2-mutant xenografts in mice, as well. We also provide further evidence that ICG-001 inhibits proliferation of NF2-mutant meningioma cells at least partly through attenuating the FOXM1-mediated Wnt/β-catenin signaling.ConclusionsThis study highlights the importance of ligand-mediated Wnt/β-catenin signaling as well as its drugable potency in NF2-mutant meningioma.

DDIS-34. HIGH-THROUGHPUT DRUG SCREENING OF FDA-APPROVED ANTINEOPLASTIC DRUGS FOR THE TREATMENT OF AGGRESSIVE MENINGIOMAS

Abstract Currently, we are facing several challenges in the treatment of meningiomas: only subtotally removable tumors, high rate of recurrence in higher-grade meningiomas, malignancy, and lack of effective chemotherapeutic agents for aggressive or inoperable tumors. For these reasons, there is an urgent need for more successful treatments for aggressive meningiomas. Therefore, we used 147 FDA-approved antineoplastic drugs on two different meningioma cell lines, including the genetically modified cell line Ben-Men-1 (WHO°I) and the newly established anaplastic meningioma cell line NCH93 (WHO°III). The impact of the drugs on proliferation was assessed in a high-throughput 386-well format with CellTiter-Glo (Promega) and further validation was done by crystal-violet assay. Subsequent screening of 147 FDA-approved drugs resulted in the identification of potential 5 drugs, including Bortezomib, Carfilzomib, Omacetaxine, Paclitaxel, and Ponatinib. Dose-curve analysis revealed IC50 values of these drugs in the Ben-Men-1 and NCH93 cell lines as follows: Bortezomib 16.2 and 5.7 nM, Carfilzomib 4.8 and 2.6 nM, Omacetaxine and 5.0 and 8.9 nM, Paclitaxel 2.6 and 1.9 µM, and Ponatinib 278 and 206 nM. Inhibitor dosage of 10xIC50 values and higher lead to an average inhibition of Ben-Men-1 and NCH93 cells by up to 90% on day 2 (P< .001). The impact of the antineoplastic agents on the cell cycle was analyzed by flow cytometry. Percentages of sub-G1 phase were significantly elevated with increasing drug concentrations of all five tested compounds (P< .001). To assess the effects of the drugs on migration scratch assay was performed. Drug concentrations of 10xIC50 values resulted in an inhibition of migration in Ben-Men-1 cells for Bortezomib, Carfilzomib, and Omacetaxine by 49%, 30%, and 23%, respectively (P< .05). In conclusion, we identified 5 promising drugs for the treatment of aggressive meningiomas by applying a high-throughput drug screening of 147 FDA-approved antineoplastic drugs.

SNHG1/miR-556-5p/TCF12 feedback loop enhances the tumorigenesis of meningioma through Wnt signaling pathway.

Meningioma, as a sort of the malignantly intracranial tumors, has captured public attention for its second-highest morbidity all over the world. Long noncoding RNAs (lncRNAs), including lncRNA SNHG1, have been well known as essential players in the development of diverse cancers. However, the biological effect and regulatory mechanism of SNHG1 have not been mentioned in meningioma. In this work, it was discovered that SNHG1 was overexpressed in meningioma cell lines. SNHG1 deficiency restrained cell growth as well as accelerated apoptosis. Then mechanism experiments demonstrated that SNHG1 functioned as the role of sponging miR-556-5p and negatively regulated miR-556-5p expression. Moreover, it was verified that TCF12 is the direct downstream target of miR-556-5p. Furthermore, SNHG1/miR-556-5p/TCF12 axis promoted cell proliferation and suppressed cell apoptosis in meningioma via activating the Wnt signaling pathway. In the end, it was confirmed that TCF12 expression was positively regulated by SNHG1, and TCF12 could promote transcription of SNHG1 through binding with the promoter region of SNHG1. In conclusion, the SNHG1/miR-556-5p/TCF12 feedback loop promotes the tumorigenesis of meningioma through the Wnt signaling pathway.

Efficient ADCC killing of meningioma by avelumab and a high-affinity natural killer cell line, haNK.

Meningiomas are the most common adult primary tumor of the central nervous system, but there are no known effective medical therapies for recurrent meningioma, particularly for World Health Organization grade II and III tumors. Meningiomas arise from the meninges, located outside the blood-brain barrier, and therefore may be directly targeted by antibody-mediated immunotherapy. We found that programmed cell death ligand 1 (PD-L1) was highly expressed in multiple human malignant meningioma cell lines and patient tumor samples. PD-L1 was targeted with the anti-PD-L1 antibody avelumab and directed natural killer cells to mediate antibody-dependent cellular cytotoxicity (ADCC) of PD-L1-expressing meningioma tumors both in vitro and in vivo. ADCC of meningioma cells was significantly increased in target cells that upregulated PD-L1 expression and, conversely, abrogated in tumor cells that were depleted of PD-L1. Additionally, the high-affinity natural killer cell line, haNK, outperformed healthy donor NK cells in meningioma ADCC. Together, these data support a clinical trial designed to target PD-L1 with avelumab and haNK cells, potentially offering a novel immunotherapeutic approach for patients with malignant meningioma.

OS8.8 Modified chromatin histone marks at noncoding elements are associated with aggressive meningioma subtype

Abstract Background: Although most meningiomas are non-malignant, there is a high recurrence rate among atypical and anaplastic (malignant) meningiomas (grades II/III). In addition, malignant meningioma usually progresses after treatment. Recently, based on DNA methylation, two subgroups of meningioma were described with recurrence-free survival differences (favorable and unfavorable). Epigenetic deregulation at distinct genomic elements such as enhancers can drive changes in gene expression and alter the transcriptional profile of the cancer cells. We seek to understand the mechanisms of meningioma recurrence and progression after initial treatment. Material and Methods: Two favorable and two unfavorable meningioma high grade tumor tissue were selected for this pilot study. Immunoprecipitation (Antibodies) for two different histone modifications (H3K4me3 and H3K27ac) were used to generate genome-wide chromatin-IP sequencing. Genome-wide DNA methylation profile was assessed for 90 meningioma cases including the four samples used in this pilot study. Results: Using a FDR cutoff of 5%, we identified 10,049 H3K27ac and 5,752 H3K4me3 chromatin marks that distinguish favorable from unfavorable meningioma. We dichotomized the results into genomic regions overlapping known gene promoters and non-promoters. Promoter associated chromatin changes (H3K27ac and H3K4me3) coincide with DNA methylation changes in aggressive meningioma. The top 1,000 H3K27ac (sorted by fold difference) without H3K4me3 coincide with highly conserved DNA elements known to be associated with enhancers. These enhancers can drive expression of multiple genes simultaneously. Using DNA methylation differences between favorable and unfavorable meningioma, we will overlap with our unfavorable associated enhancers in order to refine our candidate enhancers for follow up mechanistic studies using aggressive meningioma cell lines. Conclusion: Our preliminary results are the first to unravel the genome-wide chromatin changes associated with unfavorable or clinically aggressive meningioma. Identification of these candidate enhancers will provide knowledge of the role of epigenomics in the development of malignant meningioma and of opportunities for targeted therapy. This project is supported by the Henry Ford Health System, Department of Neurosurgery and the Hermelin Brain Tumor Center Foundation (A30935), United States National Institutes of Health (R01CA222146), and United States Department of Defense (CA170278)

Folate can promote the methionine-dependent reprogramming of glioblastoma cells towards pluripotency

Methionine dependency of tumor growth, although not well-understood, is detectable by 11C-methionine positron emission tomography and may contribute to the aggressivity of glioblastomas (GBM) and meningiomas. Cytosolic folate cycle is required for methionine synthesis. Its dysregulation may influence cell reprogramming towards pluripotency. We evaluated methionine-dependent growth of monolayer (ML) cells and stem cell-like tumor spheres (TS) derived from 4 GBM (U251, U87, LN299, T98G) and 1 meningioma (IOMM-LEE) cell lines. Our data showed that for all cell lines studied, exogenous methionine is required for TS formation but not for ML cells proliferation. Furthermore, for GBM cell lines, regardless of the addition of folate cycle substrates (folic acid and formate), the level of 3 folate isoforms, 5-methytetrahydrofolate, 5,10-methenyltetrahydrofolate, and 10-formyltetrahydrofolate, were all downregulated in TS relative to ML cells. Unlike GBM cell lines, in IOMM-LEE cells, 5-methyltetrahydrofolate was actually more elevated in TS than ML, and only 5,10-methenyltetrahydrofolate and 10-formyltetrahydrofolate were downregulated. The functional significance of this variation in folate cycle repression was revealed by the finding that Folic Acid and 5-methyltetrahydrofolate promote the growth of U251 TS but not IOMM-LEE TS. Transcriptome-wide sequencing of U251 cells revealed that DHFR, SHMT1, and MTHFD1 were downregulated in TS vs ML, in concordance with the low activity cytosolic folate cycle observed in U251 TS. In conclusion, we found that a repressed cytosolic folate cycle underlies the methionine dependency of GBM and meningioma cell lines and that 5-methyltetrahydrofolate is a key metabolic switch for glioblastoma TS formation. The finding that folic acid facilitates TS formation, although requiring further validation in diseased human tissues, incites to investigate whether excessive folate intake could promote cancer stem cells formation in GBM patients.

Forkhead box M1 (FOXM1) transcription factor is a key oncogenic driver of aggressive human meningioma progression.

Aggressive meningioma remains incurable with neither chemo- nor targeted therapies proven effective, largely due to unidentified genetic alterations and/or aberrant oncogenic pathways driving the disease progression. In this study, we examined the expression and function of Forkhead box M1 (FOXM1) transcription factor during meningioma progression. Human meningioma samples (n=101) were collected, followed by Western blotting, quantitative PCR, immunohistochemical and progression-free survival (PFS) analyses. For in vitro assays, FOXM1 was overexpressed or knocked-down in benign (SF4433 and SF4068) or malignant (SF3061 and IOMM-Lee) human meningioma cell lines respectively. For in vivo studies, siomycin A (a FOXM1 inhibitor)-pretreated or control IOMM-Lee cells were implanted subcutaneously in nude mice. FOXM1 expression was increased in higher grades of meningioma and correlated with the mitotic index in the tumour tissue. Moreover, FOXM1 was increased in recurrent meningioma compared with the matched primary lesions. The patients who had higher FOXM1 expression had shorter PFS. In the subsequent in vitro assays, knockdown of FOXM1 in malignant meningioma cell lines resulted in decreased tumour cell proliferation, angiogenesis and invasion, potentially via regulation of β-catenin, cyclin D1, p21, interleukin-8, vascular endothelial growth factor-A, PLAU, and epithelial-to-mesenchymal transition-related genes, whereas overexpression of FOXM1 in benign meningioma cell lines had the opposite effects. Last, suppression of FOXM1 using a pharmacological inhibitor, siomycin A, decreased tumour growth in an in vivo mouse model. Our data demonstrate that FOXM1 is a key transcription factor regulating oncogenic signalling pathways in meningioma progression, and a promising therapeutic target for aggressive meningioma.

Antitumor Effects of Chrysophanol in Malignant Optic Nerve Meningioma Cell Lines are Mediated via Caspase Activation, Induction of Mitochondrial Mediated Apoptosis, Mitochondrial Membrane Depolarization and Targeting the Mitogen-Activated Protein Kinase Signaling Pathway

Anthroquinones are considered remarkable anticancer agents. Chrysophanol is an important anthroquinone and it has shown to have the potential to inhibit the growth of the range of cancers. However, there are no studies regarding the anticancer effects of chrysophanol against the malignant meningioma of optic nerve. In this review, the potential of chrysophanol in the treatment of malignant -meningioma of optic nerve was explored by evaluating its anticancer activity against the malignant meningioma CH157-MN cells. The 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay was used for cell viability determination. The 4',6-diamidino-2-phenylindole (DAPI), acridine orange and ethidium bromide (AO/EB) and annexin V/PI assays were used to determine the induction of apoptosis. The potential of reactive oxygen species and the mitochondrial membrane was estimated by flow cytometry. Western blot analysis was performed to determine the protein expression. The results showed that chrysophanol caused significant decline in the viability of the CH157-MN cells and exhibited an IC50 of 30 µmol/L. Anticancer effects were found to be due to the induction of apoptosis as evident form the DAPI and AO/EB staining. The annexin V/PI staining revealed that the apoptotic cells increased from 1.77% in control to 37.21% at 60 µmol/L concentration of chrysophanol. The Bcl-2/Bax expression ratio was decreased and the caspases-3 and 9 were activated upon chrysophanol treatment of the CH157-MN cells. Chrysophanol also triggered the formation of reactive oxygen species and reduction of the mitochondrial membrane potential in the CH157-MN cells and also blocked the Mitogen-activated protein kinase signaling pathway. The findings of the present study suggest that chrysophanol may prove beneficial in the treatment of malignant meningioma of optic nerve. Key Message: The study revealed the anticancer potential of chrysophanol against the malignant optic nerve meningioma.

Identification of KIF11 As a Novel Target in Meningioma.

Kinesins play an important role in many physiological functions including intracellular vesicle transport and mitosis. The emerging role of kinesins in different cancers led us to investigate the expression and functional role of kinesins in meningioma. Therefore, we re-analyzed our previous microarray dataset of benign, atypical, and anaplastic meningiomas (n = 62) and got evidence for differential expression of five kinesins (KIFC1, KIF4A, KIF11, KIF14 and KIF20A). Further validation in an extended study sample (n = 208) revealed a significant upregulation of these genes in WHO°I to °III meningiomas (WHO°I n = 61, WHO°II n = 88, and WHO°III n = 59), which was most pronounced in clinically more aggressive tumors of the same WHO grade. Immunohistochemical staining confirmed a WHO grade-associated upregulated protein expression in meningioma tissues. Furthermore, high mRNA expression levels of KIFC1, KIF11, KIF14 and KIF20A were associated with shorter progression-free survival. On a functional level, knockdown of kinesins in Ben-Men-1 cells and in the newly established anaplastic meningioma cell line NCH93 resulted in a significantly inhibited tumor cell proliferation upon siRNA-mediated downregulation of KIF11 in both cell lines by up to 95% and 71%, respectively. Taken together, in this study we were able to identify the prognostic and functional role of several kinesin family members of which KIF11 exhibits the most promising properties as a novel prognostic marker and therapeutic target, which may offer new treatment options for aggressive meningiomas.

LINC00702/miR-4652-3p/ZEB1 axis promotes the progression of malignant meningioma through activating Wnt/β-catenin pathway

Long noncoding RNAs (lncRNAs) have been acknowledged as significant regulators in the progression of various cancers, including malignant meningioma. Being a newly identified lncRNA, long intergenic non-protein coding RNA 702 (LINC00702) has not been comprehensively studied in malignant meningioma. In this study, the role of LINC00702 was identified and explored in malignant meningioma. The LINC00702 expression was determined in malignant meningioma tissues and cell lines by qRT-PCR. Then the association between LINC00702 expression and the prognosis of malignant meningioma patients was analyzed by Kaplan-Meier analysis. The functional role of LINC00702 in malignant meningioma cell proliferation and migration was analyzed by loss-of function assays. Subcellular fractionation assay and FISH assay revealed the cytoplasmic localization of LINC00702. Bioinformatics analysis and mechanism experiments demonstrated that LINC00702 acted as the molecular sponge of miR-4652-3p to upregulate ZEB1 in malignant meningioma. Furthermore, high level of LINC00702 was demonstrated to be associated with the activity of Wnt/β-catenin signaling. Moreover, miR-4652-3p and ZEB1 involved in LINC00702-mediated Wnt/β-catenin signaling. At last, rescue assays revealed that miR-4652-3p and ZEB1 attenuated LINC00702-mediated cell proliferation and migration.

Immunohistochemical Characterization of Procaspase-3 Overexpression as a Druggable Target With PAC-1, a Procaspase-3 Activator, in Canine and Human Brain Cancers.

Gliomas and meningiomas are the most common brain neoplasms affecting both humans and canines, and identifying druggable targets conserved across multiple brain cancer histologies and comparative species could broadly improve treatment outcomes. While satisfactory cure rates for low grade, non-invasive brain cancers are achievable with conventional therapies including surgery and radiation, the management of non-resectable or recurrent brain tumors remains problematic and necessitates the discovery of novel therapies that could be accelerated through a comparative approach, such as the inclusion of pet dogs with naturally-occurring brain cancers. Evidence supports procaspase-3 as a druggable brain cancer target with PAC-1, a pro-apoptotic, small molecule activator of procaspase-3 that crosses the blood-brain barrier. Procaspase-3 is frequently overexpressed in malignantly transformed tissues and provides a preferential target for inducing cancer cell apoptosis. While preliminary evidence supports procaspase-3 as a viable target in preclinical models, with PAC-1 demonstrating activity in rodent models and dogs with spontaneous brain tumors, the broader applicability of procaspase-3 as a target in human brain cancers, as well as the comparability of procaspase-3 expressions between differing species, requires further investigation. As such, a large-scale validation of procaspase-3 as a druggable target was undertaken across 651 human and canine brain tumors. Relative to normal brain tissues, procaspase-3 was overexpressed in histologically diverse cancerous brain tissues, supporting procaspase-3 as a broad and conserved therapeutic target. Additionally, procaspase-3 expressing glioma and meningioma cell lines were sensitive to the apoptotic effects of PAC-1 at biologically relevant exposures achievable in cancer patients. Importantly, the clinical relevance of procaspase-3 as a potential prognostic variable was demonstrated in human astrocytomas of variable histologic grades and associated clinical outcomes, whereby tumoral procaspase-3 expression was negatively correlated with survival; findings which suggest that PAC-1 might provide the greatest benefit for patients with the most guarded prognoses.

Telomerase activity, TERT expression, hTERT promoter alterations, and alternative lengthening of the telomeres (ALT) in meningiomas - a systematic review.

Telomerase activity and (human) Telomerase Reverse Transcriptase (hTERT) expression are considered hallmarks in oncogenesis of neoplasms and are upregulated by alterations of the hTERT promoter. In meningiomas, numerous studies investigated hTERT expression, telomerase activity, promoter mutations, and methylations. Moreover, reports about hTERT-targeted chemotherapy in meningiomas have recently been published. We provide a systematic review of the literature about the role of hTERT in meningiomas. TERT expression and telomerase activity is found in benign and high-grade meningiomas and increase with WHO grade. Remarkably, rates of TERT expression/telomerase activity usually exceed mutation frequency and both telomerase activity and TERT expression have also been found in hTERT promoter wildtype meningiomas, indicating further mechanisms of TERT upregulation. Although hTERT promoter methylation has been reported in the vast majority of meningiomas, correlation with TERT expression remains controversial. Rates of promoter mutations, and methylation were shown to increase with rising WHO grade. Moreover, promoter methylation and mutations strongly correlate with prognosis. Although mutations predicted malignant progression, de novo mutations in high-grade recurrences of former benign lesions were also observed. Retroviral transduction of the TERT gene enabled immortalization in several grade I-III meningioma cell lines. In vitro analyses revealed significant effects on viability in hTERT-mutated meningioma cells after targeted treatment. Alternative mechanisms of telomere lengthening are usually absent in meningiomas. TERT and hTERT promoter alterations play a major role during oncogenesis of meningiomas with implications for prognosis and potentially treatment.

Mebendazole and radiation in combination increase survival through anticancer mechanisms in an intracranial rodent model of malignant meningioma

Meningiomas are a frequent tumor of the central nervous system. Although mostly benign, approximately 5% present as atypical or malignant tumors. Treatments for atypical meningiomas include gross total resection and radiotherapy, but about 33% of patients have recurrent tumors, sometimes as a higher grade. Recently, the brain penetrant anthelmintic drug, mebendazole, has shown promise as an anticancer agent in rodent models of glioblastoma and medulloblastoma. The half maximal inhibitory concentration (IC50) effect on colony formation, cell proliferation, and caspase-3/7 markers of apoptosis of mebendazole with and without radiation was measured in vitro. Mice intracranially implanted with KT21MG1 human meningioma were administered mebendazole alone or in combination with radiation. Survival benefit was evaluated, while tumors were investigated by immunohistochemical staining for apoptosis, cell proliferation, and vascular density. In vitro experiments on meningioma cell lines showed the IC50 for mebendazole in the range of 0.26-0.42µM. Mebendazole alone induced cytotoxicity, however the combination had a greater reduction in colony formation and resulted in higher levels of cleaved caspase-3. The in vivo study showed both, mebendazole alone and the combination, to have a survival benefit with an increase in apoptosis, and decreases in tumor cell and vascular proliferation. These preclinical findings indicate that mebendazole alone or in combination with radiation can be considered for the treatment of malignant meningioma. The mechanism of action for this combination may include an increase in apoptosis, a reduction in proliferation and angiogenesis, or a combination of these effects.

Photodynamic therapy with talaporfin sodium induces dose- and time-dependent apoptotic cell death in malignant meningioma HKBMM cells.

To investigate the effect of photodynamic therapy (PDT) with the talaporfin sodium (mono-L-asparthyl chlorine e6: NPe-6) on human malignant meningioma cell line HKBMM cells in vitro. After incubation with NPe6 for 4 h, cells underwent PDT (diode laser irradiation: 3.4 mW/cm2 and 1 J/cm2. Cell viability was determined in 2 malignant meningioma cell lines (human origin; HKBMM cells and rat origin; KMY-J cells) and human malignant glioma U251 cells with Cell Counting Kit-8 assay. The HKBMM cells were examined for caspase-3 activity, annexin V or propidium iodide (PI) staining, and lactate dehydrogenase leakage. Morphological change was also investigated with phase-contrast microscopy. In human malignant meningioma HKBMM cells, viability showed a dose- and time-dependent decrease. After 24 h of laser irradiation, NPe6 at 20 μg/ml or more induced a significant decrease in cell viability in both HKBMM cells and KMY-J cells, although they more resistance than the malignant glioma cell line U251 cells. Two kinds of morphological change were also observed in the HKBMM cells, shrinkage of the cell body, indicating apoptosis, and swelling of the cell body, indicating necrosis. In addition, both caspase-3 activity and DNA fragmentation, biochemical markers indicative of apoptosis, showed a dose-dependent increase. The percentage of necrotic cells showing positive staining for annexin V or PI was greater than that of apoptotic cells at a high concentration of NPe6. Lactate dehydrogenase leakage, a biochemical marker of necrosis, also showed a marked increase at a high concentration of NPe6. Photodynamic therapy with NPe6 induced dose- and time-dependent apoptosis in human malignant meningioma HKBMM cells. At a high concentration of NPe6, however, it induced necrosis.

Abstract 4398: In vitro Tumor Treating Fields (TTFields) alter proliferation and morphology of patient-derived high-grade meningioma cell lines

Abstract Introduction: Meningiomas are the most common neoplasm of the central nervous system (CNS), accounting for 37% of primary CNS tumors. Though derived from the arachnoid cap cells within the meninges, high-grade meningiomas (WHO grades II and III) invade into the normal brain tissue, and have a high rate of recurrence. As there are no efficacious chemotherapies for these high-grade meningiomas, standard of care is currently limited to maximal surgical resection followed by radiotherapy, with a median overall survival of only 2.5-3 years. Clinical trials with TTFields therapy are under way for patients with high-grade meningiomas. In this study, in vitro experiments comparing TTFields to untreated controls were performed on two patient-derived anaplastic (WHO grade III) meningioma cell lines to determine the effects of TTFields on cell proliferation and clonogenicity. Methods: Studies utilizing patient tumor specimens were approved by the Wayne State University Institutional Review Board and written informed consent was obtained from participants. Meningioma tumor specimens were collected immediately following microsurgical resection. Single-cell suspensions from the tumor tissues were prepared by enzymatic and mechanical disruption. Cells were plated on plastic coverslips (10,000 each) in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at ~1.6 V/cm at 100, 200, 300, or 400 kHz (groups n=4-5) with one control group. Culture media were replaced every day. After 12 days of treatment, cell proliferation was assessed with the XTT assay and cells were harvested and replated for clonogenic assays (500 cells/well), which were stained with crystal violet and counted with an automated colony counter. Harvested cells were also fixed and immunostained with the diagnostic meningioma cytoplasmic marker vimentin to observe the cell morphology in control vs. TTFields-treated cells. Control vs. TTFields-treated groups were compared by one- or two-tailed t-tests. Results: The 12-day TTFields treatment significantly reduced cell proliferation, with the greatest decrease found in the 200 kHz group (0.508±0.21; mean±SEM) compared to the control (1.515±0.08; two-tailed t test p=0.0019). Similar results were found in the clonogenic assay, with control cell counts of 4219±793 compared to the 200kHz treated cells 1793±444 (one-tailed t test p=0.0279). In the vimentin-stained cells, the control cells displayed a typical meningioma spindle morphology, while the TTFields-treated cells demonstrated pleomorphic and irregular morphologies. Conclusions: Application of TTFields in vitro reduced proliferation and clonogenicity and altered morphology of patient-derived anaplastic meningioma cell lines. This is the first report on the in vitro effects of TTFields on meningioma. Citation Format: Sharon K. Michelhaugh, Sam Kiousis, Sam A. Michelhaugh, Neil V. Klinger, Sandeep Mittal. In vitro Tumor Treating Fields (TTFields) alter proliferation and morphology of patient-derived high-grade meningioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4398.