Can permanent cell lines originate from low-grade astrocytomas: overgrowth of “glia-like“ cells in pilocytic and fibrillary (diffuse) astrocytoma cultures

Abstract Current treatment for diffuse astrocytoma fails to address its underlying molecular mechanisms leading to inevitable disease progression and eventual patient death. Genomic studies have implicated ATRX alterations as critical to low grade glioma biology. Our lab has previously shown in vitro that ATRX influences glioma motility, cellular differentiation state, and epigenetic programming, however, the influence of ATRX alterations in vivo remains unclear. Here, we leveraged an RCAS/tva mouse tumor model to probe the role of ATRX deficiency in glioma. Atrx deficient murine tumors exhibited lower histopathological grade and were associated with longer survival than Atrx-intact counterparts, and syngeneic allografts of cell lines derived from primary tumors mirrored the differential degrees of aggressiveness seen in primary tumors. Tumor-derived Atrx-deficient cell lines showed increased susceptibility to G-quadruplex stabilizing compounds, pointing to increased replication stress and recapitulating a key phenotype of ATRX-mutant gliomas in humans. Transcriptional profiling revealed enrichments in MYC target genes, E2F targets as well as G2/M checkpoint pathways in Atrx-intact tumors and cells, and enrichment in RAS signaling in Atrx-deficient tumors and cells. Finally, Atrx deficient murine gliomas displayed increased levels of NK cells, a phenotype recapitulated in ATRX-mutant human gliomas, and primary Atrx-deficient glioma lines exhibited increased levels of NK cell-attracting cytokines. These latter findings suggest that ATRX deficiency could influence interactions between glioma cells and their immune microenvironment by way of phenotypically relevant molecular mechanisms.

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Molecular Landscape for Malignant Transformation in Diffuse Astrocytoma

Global Medical Genetics ◽

10.1055/s-0041-1731069 ◽

2021 ◽

Author(s):

Thara Tunthanathip ◽

Surasak Sangkhathat ◽

Kanet Kanjanapradit

Keyword(s):

Malignant Transformation ◽

Extent Of Resection ◽

Low Grade ◽

Diffuse Astrocytoma ◽

Continuous Variables ◽

Isocitrate Dehydrogenase 1 ◽

Molecular Alteration ◽

Exact Test ◽

Molecular Abnormalities ◽

Biological Variables

Abstract Background Malignant transformation (MT) of low-grade gliomas changes dramatically the natural history to poor prognosis. Currently, factors associated with MT of gliomas have been inconclusive, in particular, diffuse astrocytoma (DA). Objective The present study aims to explore the molecular abnormalities related to MT in the same patients with different MT stages. Methods Twelve specimens from five DA patients with MT were genotyped using next-generation sequencing (NGS) to identify somatic variants in different stages of MT. We used cross-tabulated categorical biological variables and compared the mean of continuous variables to assess for association with MT. Results Ten samples succussed to perform NGS from one male and four females, with ages ranging from 28 to 58 years. The extent of resection was commonly a partial resection following postoperative temozolomide with radiotherapy in 25% of cases. For molecular findings, poly-T-nucleotide insertion in isocitrate dehydrogenase 1 (IDH1) was significantly related to MT as a dose–response relationship (Mann–Whitney's U test, p = 0.02). Also, mutations of KMT2C and GGT1 were frequently found in the present cohort, but those did not significantly differ between the two groups using Fisher's exact test. Conclusion In summary, we have identified a novel relationship between poly-T insertion polymorphisms that established the pathogenesis of MT in DA. A further study should be performed to confirm the molecular alteration with more patients.

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DDRE-03. IDH1-MUTANT GBM CELLS ARE HIGHLY SENSITIVE TO COMBINATION OF KDM6A/B AND HDAC INHIBITORS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab024.025 ◽

2020 ◽

Vol 3 (Supplement_1) ◽

pp. i6-i7

Author(s):

Alişan Kayabölen ◽

Gizem Nur Sahin ◽

Fidan Seker ◽

Ahmet Cingöz ◽

Bekir Isik ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Mutant Cell ◽

Glioma Cells ◽

Epigenetic Therapy ◽

Low Grade ◽

Orthotopic Model ◽

Cycle Arrest ◽

Mutant Cells

Abstract Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

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HGG-02. ADOLESCENT AND YOUNG ADULT (AYA) GLIOMA WITH BRAF V600E-MUTANTATION

Neuro-Oncology ◽

10.1093/neuonc/noaa222.294 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii344-iii344

Author(s):

Yui Kimura ◽

Yukitomo Ishi ◽

Yuko Watanabe ◽

Yoshiko Nakano ◽

Shigeru Yamaguchi ◽

...

Keyword(s):

Braf Inhibitor ◽

Clinical Information ◽

Early Recurrence ◽

Pleomorphic Xanthoastrocytoma ◽

Adolescent And Young Adult ◽

Braf V600e ◽

Low Grade ◽

Diffuse Astrocytoma ◽

Genetic Features ◽

Epithelioid Glioblastoma

Abstract BACKGROUND Biological features of pediatric glioma differ significantly from those of adult glioma, and limited data are available on those of AYA patients. Here, we focused on AYA patients with glioma, especially those harboring BRAF V600E mutation, and investigated their clinical and genetic features. METHOD: We retrospectively analyzed AYA patients with brain tumors harboring BRAF V600E, who were treated in two hospitals in Japan. RESULTS Clinical information was available for 14 patients. The median age at diagnosis was 25 years (range: 15–38). Five patients were diagnosed with glioblastoma (GBM), including one epithelioid type. These patients were over 25. Although one patient with GBM died of the disease 6.9 years after initial diagnosis, the remaining patients were alive. Two patients were alive without recurrence at 38 and 51 months after the treatment. The patient with epithelioid glioblastoma experienced early recurrence. The remaining nine patients (64%) were diagnosed with low-grade glioma, including ganglioglioma, pilocytic astrocytoma, diffuse astrocytoma, oligodendroglioma, pleomorphic xanthoastrocytoma, and polymorphous low-grade neuroepithelial tumor of the young. No patients died of the disease, and four patients are alive without recurrence after initial operation without adjuvant treatment. Two patients are (epithelioid glioblastoma and ganglioglioma) currently undergoing treatment with a BRAF inhibitor for recurrent tumors. DISCUSSION Although the number of this study is limited, our study suggested that the prognosis of AYA patients with BRAF-V600E positive GBM may not be as dismal as that of children or adults.

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The Interplay between Oxidative Phosphorylation and Glycolysis as a Potential Marker of Bladder Cancer Progression

International Journal of Molecular Sciences ◽

10.3390/ijms21218107 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8107 ◽

Cited By ~ 1

Author(s):

Greta Petrella ◽

Giorgia Ciufolini ◽

Riccardo Vago ◽

Daniel Oscar Cicero

Keyword(s):

Bladder Cancer ◽

Oxidative Phosphorylation ◽

Cell Lines ◽

Cancer Progression ◽

Cancer Genomics ◽

Low Grade ◽

Metabolic State ◽

Extracellular Medium ◽

Potential Marker ◽

Risk Of Progression

Urothelial bladder cancer (UBC) is the most common tumor of the urinary system. One of the biggest problems related to this disease is the lack of markers that can anticipate the progression of the cancer. Genomics and transcriptomics have greatly improved the prediction of risk of recurrence and progression. Further progress can be expected including information from other omics sciences such as metabolomics. In this study, we used 1H-NMR to characterize the intake of nutrients and the excretion of products in the extracellular medium of three UBC cell lines, which are representatives of low-grade tumors, RT4, high-grade, 5637, and a cell line that shares genotypic features with both, RT112. We have observed that RT4 cells show an activated oxidative phosphorylation, 5637 cells depend mostly on glycolysis to grow, while RT112 cells show a mixed metabolic state. Our results reveal the relative importance of glycolysis and oxidative phosphorylation in the growth and maintenance of different UBC cell lines, and the relationship with their genomic signatures. They suggest that cell lines associated with a low risk of progression present an activated oxidative metabolic state, while those associated with a high risk present a non-oxidative state and high glycolytic activity.

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Human Squamous Cell Carcinoma: Establishment and Characterization of New Permanent Cell Lines

Archives of Otolaryngology - Head and Neck Surgery ◽

10.1001/archotol.1981.00790470051012 ◽

1981 ◽

Vol 107 (11) ◽

pp. 703-710 ◽

Cited By ~ 105

Author(s):

C. J. Krause ◽

T. E. Carey ◽

R. W. Ott ◽

C. Hurbis ◽

K. D. McClatchey ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Cell Lines ◽

Squamous Cell ◽

Human Squamous Cell Carcinoma ◽

Permanent Cell

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LGG-17. SYNERGISTIC ACTIVITY OF MAPK INHIBITOR CLASSES REVEALED BY A NOVEL CELL-BASED MAPK ACTIVITY PEDIATRIC LOW-GRADE GLIOMA ASSAY

Neuro-Oncology ◽

10.1093/neuonc/noaa222.399 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii369-iii369

Author(s):

Diren Usta ◽

Romain Sigaud ◽

Juliane L Buhl ◽

Florian Selt ◽

Viktoria Marquardt ◽

...

Keyword(s):

Cell Lines ◽

Mapk Pathway ◽

Braf V600e ◽

Vector System ◽

Luciferase Reporter ◽

Low Grade ◽

Brafv600e Mutation ◽

Pathway Activity ◽

Combination Treatments ◽

Braf Fusion

Abstract Pilocytic astrocytomas (PAs) and other pediatric low-grade gliomas (pLGGs) exhibit aberrant activation of the MAPK signaling pathway caused by genetic alterations, most commonly KIAA1549:BRAF fusions, BRAF V600E and NF1 mutations. In such a single-pathway disease, novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for treatment. We developed an assay suitable for pre-clinical testing of MAPKi in pLGGs, aiming at the identification of novel MAPK pathway suppressing synergistic drug combinations. We generated a reporter plasmid (pDIPZ) expressing destabilized firefly luciferase driven by a MAPK-responsive ELK-1-binding element, packaged in a lentiviral vector system. We stably transfected pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively. Measurement of MAPK pathway activity was performed using the luciferase reporter. pERK protein levels were detected for validation. We performed a screen of a MAPKi library and calculated Combination Indices of selected combinations. The MAPKi library screen revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and second generation RAF inhibitors. Synergistic effects in both BRAF-fusion and BRAFV600E mutation backgrounds were observed following combination treatments with different MAPKi classes (RAFi/MEKi, > RAFi/ERKi > MEKi/ERKi). We have generated a novel reporter assay for medium- to high-throughput pre-clinical drug testing of MAPKi in pLGG cell lines. MEK, ERK and next-generation RAF inhibitors were confirmed as potential treatment approaches for KIAA1549:BRAF and BRAFV600E mutated pLGGs. Synergistic suppression of MAPK pathway activity upon combination treatments was revealed using our assay in addition.

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Molecular Alterations in Pediatric Low-Grade Gliomas That Led to Death

Journal of Neuropathology & Experimental Neurology ◽

10.1093/jnen/nlab097 ◽

2021 ◽

Author(s):

Jared T Ahrendsen ◽

Claire Sinai ◽

David M Meredith ◽

Seth W Malinowski ◽

Tabitha M Cooney ◽

...

Keyword(s):

Braf V600e ◽

Molecular Characteristics ◽

Low Grade ◽

Diffuse Astrocytoma ◽

Term Survival ◽

Pten Loss ◽

Low Grade Gliomas ◽

Molecular Alterations ◽

Idh1 R132h ◽

Poor Outcomes

Abstract Pediatric low-grade gliomas (PLGGs) have excellent long-term survival, but death can occasionally occur. We reviewed all PLGG-related deaths between 1975 and 2019 at our institution: 48 patients were identified; clinical data and histology were reviewed; targeted exome sequencing was performed on available material. The median age at diagnosis was 5.2 years (0.4–23.4 years), at death was 13.0 years (1.9–43.2 years), and the overall survival was 7.2 years (0.0–33.3 years). Tumors were located throughout CNS, but predominantly in the diencephalon. Diagnoses included low-grade glioma, not otherwise specified (n = 25), pilocytic astrocytoma (n = 15), diffuse astrocytoma (n = 3), ganglioglioma (n = 3), and pilomyxoid astrocytoma (n = 2). Recurrence occurred in 42/48 cases, whereas progression occurred in 10. The cause of death was direct tumor involvement in 31/48 cases. Recurrent drivers included KIAA1549-BRAF (n = 13), BRAF(V600E) (n = 3), NF1 mutation (n = 3), EGFR mutation (n = 3), and FGFR1-TACC1 fusion (n = 2). Single cases were identified with IDH1(R132H), FGFR1(K656E), FGFR1 ITD, FGFR3 gain, PDGFRA amplification, and mismatch repair alteration. CDKN2A/B, CDKN2C, and PTEN loss was recurrent. Patients who received only chemotherapy had worse survival compared with patients who received radiation and chemotherapy. This study demonstrates that PLGG that led to death have diverse molecular characteristics. Location and co-occurring molecular alterations with malignant potential can predict poor outcomes.

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Glucose metabolism by adult hepatocytes in primary culture and by cell lines from rat liver

AJP Cell Physiology ◽

10.1152/ajpcell.1978.234.3.c122 ◽

1978 ◽

Vol 234 (3) ◽

pp. C122-C130 ◽

Cited By ~ 70

Author(s):

D. M. Bissell ◽

G. A. Levine ◽

M. J. Bissell

Keyword(s):

Glucose Metabolism ◽

Primary Culture ◽

Cell Lines ◽

Rat Liver ◽

Liver Cell ◽

Time Course ◽

Primary Cultures ◽

Functional Change ◽

Permanent Cell ◽

Rat Hepatoma

The metabolic fate of [U-14C]glucose has been examined in detail in adult rat hepatocytes in primary monolayer culture, as well as in two permanent cell lines--Buffalo rat liver (BRL) and transplantable rat hepatoma (HTC) cells-derived from normal rat liver and from rat hepatoma, respectively. Under defined conditions of incubation, at a glucose concentration of 5.5 mM, the three types of cultured liver cells exhibited pronounced differences in glucose metabolism. Primary cultures, like the intact liver, differed from the cell lines in consuming relatively small amounts of glucose and converting approximately 50% of the total metabolized glucose to lactate. By contrast, the permantent cell lines consumed glucose at a 40-fold greater rate than did primary cultures, converting 80--90% of the carbohydrate to lactate. Oxidative metabolism of glucose carbon also differed among the three types of liver culture. Of the total [U-14C]glucose consumed, primary cultures converted approximately 30% to labeled CO2 per hour, whereas the liver cell lines converted 5--10%. Finally, glucose metabolism in primary culture exhibited adaptation as hepatocytes aged in culture, shifting progressively toward the pattern exhibited by the permanent cell lines. This change occurred over a time course similar to that for other kinds of functional change in hepatocytes in primary culture and thus may be relevant to the general problem of phenotypic alteration in liver cell culture.

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Preferential expression of double-stranded ribonucleic acid in tumor versus normal cells: biological and clinical implications

Blood ◽

10.1182/blood.v66.1.39.bloodjournal66139 ◽

1985 ◽

Vol 66 (1) ◽

pp. 39-46

Author(s):

HM Kantarjian ◽

B Barlogie ◽

M Pershouse ◽

D Swartzendruber ◽

MJ Keating ◽

...

Keyword(s):

Tumor Cells ◽

Cell Lines ◽

Ribonucleic Acid ◽

Flow Cytometric Analysis ◽

Myelogenous Leukemia ◽

Cycle Phase ◽

Low Grade ◽

Normal Cells ◽

Normal Peripheral Blood ◽

Rna Content

In an effort to develop a new tumor marker suitable for flow cytometric analysis, we examined the value of double-stranded ribonucleic acid (ds- RNA) measurements using propidium iodide after DN'ase treatment. Cellular ds-RNA content was evaluated both in experimental cell lines and in clinical specimens. Higher levels of ds-RNA were present in tumor cells as compared with normal cells. In tumor cells, fluorescence was intensely localized in the nucleolus and was more diffuse in the cytoplasm. Change of less than 10% in the ds-RNA levels was observed in cell lines as a function of cytokinetic determinants such as cycle phase, culture age, and cycle traverse rate. Tumor differentiation by dimethylsulfoxide resulted in a significant decrease in cellular ds-RNA content. For quantitative comparison of clinical material, a ds-RNA excess was defined in relationship to normal peripheral blood lymphocytes. ds-RNA excess greater than 30% was observed in only one of 34 normal tissues (3%) as compared with 124 of 201 neoplastic tissue samples (62%). This incidence was higher in patients with acute leukemia (76%), high-grade and intermediate-grade lymphoma (75%), and high tumor stage myeloma (83%), as compared with chronic leukemia (20%), low-grade lymphoma (25%), and intermediate or low tumor mass myeloma (43%). Prognostically, a high pretreatment ds-RNA excess in myeloma was associated with a lower remission rate. The persistence of ds-RNA excess in the bone marrow of patients with acute myelogenous leukemia in remission predicted for a shorter remission duration (seven v 22 months; P = .05). We conclude that ds-RNA excess, as readily measured objectively and quantitatively by flow cytometry, may have important diagnostic and prognostic implications for the management of patients with malignant disease.

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