scholarly journals PDTM-26. NOVEL SHARED AND DISTINCT EPIGENOMIC MECHANISMS OF G34R AND K27M MUTATIONS IN CHILDHOOD GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi192-vi193
Author(s):  
Michael Chen ◽  
Kelly Bush ◽  
Nichole Lewis ◽  
Vanessa Cervantes ◽  
Paul Knoepfler
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Point Mutations ◽  
Glioma Cells ◽  
Driver Mutations ◽  
High Grade ◽  
Shared Key ◽  
Induced Changes ◽  
Open Question

Abstract Alterations in histone H3.3 are common driver mutations in high-grade pediatric gliomas, but the central oncogenic mechanisms remain an open question. To identify important mutant H3.3 effectors, we used CRISPR-Cas9 to precisely introduce H3.3 K27M and G34R mutations into previously H3.3-wildtype human astrocyte and glioma cells, while in parallel reverting mutations in glioma cells back to wildtype. K27M and G34R mutations invoked some strikingly similar epigenomic effects supporting a new model in which some major aspects of their oncogenic functions are shared. For instance, both K27M and G34R induced changes at many of the same genomic loci in specific histone marks, with the largest changes in H3K27me3 including in particular within super-enhancers, which also exhibited perturbed transcriptional function. K27M and G34R mutations induced some gene expression changes that were unique to each mutation, but both mutations changed similar functional ontological clusters and ASCL1 is a shared key putative effector. H3.3 mutant glioma cells are sensitive to ASCL1 knockdown or overexpression, resulting in cell viability that is reduced or increased, respectively. Comparison of our panel of glioma cells gene-edited with precise point mutations to edited glioma cells in other studies that performed gene knockout or overexpression reveals striking differences in the resulting phenotypes. We also determined that certain drugs exhibited specificity to H3.3 mutation-bearing cells including DAPT, JQ1, and ONC201. In vivo, we found that reversion of K27M to WT in glioma cells significantly reduced tumorigenicity in mouse xenograft assays and introduction of G34R mutations in previously WT glioma cells increased tumor growth. Overall, gene editing of gliomas and comparison of otherwise isogenic sets of cells defines both distinct and shared gliomagenesis mechanisms that can be targeted for development of oncohistone-based therapeutics.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

scholarly journals Study of the biodistribution of fluorescein in glioma-infiltrated mouse brain and histopathological correlation of intraoperative findings in high-grade gliomas resected under fluorescein fluorescence guidance

2015 ◽  
Vol 122 (6) ◽  
pp. 1360-1369 ◽  
Author(s):  
Roberto Jose Diaz ◽  
Roberto Rey Dios ◽  
Eyas M. Hattab ◽  
Kelly Burrell ◽  
Patricia Rakopoulos ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Low Dose ◽  
Glioma Cells ◽  
High Grade ◽  
Fluorescein Sodium ◽  
Tumor Margins ◽  
Histopathological Correlation ◽  
High Grade Gliomas

OBJECT Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection (GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood. METHODS The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation. RESULTS The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell–specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI–guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI. CONCLUSIONS Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions.

scholarly journals Integrated computational and experimental identification of p53, KRAS and VHL mutant selection associated with CRISPR-Cas9 editing

2018 ◽  
Author(s):  
Sanju Sinha ◽  
Karina Barbosa Guerra ◽  
Kuoyuan Cheng ◽  
Mark DM Leiserson ◽  
David M Wilson ◽  
...  
Keyword(s):  
Gene Editing ◽  
Mutant Cell ◽  
Cell Types ◽  
Driver Mutations ◽  
Mutant Selection ◽  
Cancer Driver ◽  
Genome Wide ◽  
A Genome ◽  
Induced Changes ◽  
Different Cell Types

AbstractRecent studies have reported that CRISPR-Cas9 gene editing induces a p53-dependent DNA damage response in primary cells, which may select for cells with oncogenic p53 mutations11,12. It is unclear whether these CRISPR-induced changes are applicable to different cell types, and whether CRISPR gene editing may select for other oncogenic mutations. Addressing these questions, we analyzed genome-wide CRISPR and RNAi screens to systematically chart the mutation selection potential of CRISPR knockouts across the whole exome. Our analysis suggests that CRISPR gene editing can select for mutants of KRAS and VHL, at a level comparable to that reported for p53. These predictions were further validated in a genome-wide manner by analyzing independent CRISPR screens and patients’ tumor data. Finally, we performed a new set of pooled and arrayed CRISPR screens to evaluate the competition between CRISPR-edited isogenic p53 WT and mutant cell lines, which further validated our predictions. In summary, our study systematically charts and points to the potential selection of specific cancer driver mutations during CRISPR-Cas9 gene editing.

scholarly journals H3.3K27M Mutation Promotes The Migration and Invasion of Glioma Cells By Activating β-Catenin/USP1 Signaling

2022 ◽  
Author(s):  
Zhiyuan Sun ◽  
Yufu Zhu ◽  
Xia Feng ◽  
Xiaoyun Liu ◽  
Kunlin Zhou ◽  
...  
Keyword(s):  
Glioma Cells ◽  
High Grade Glioma ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Migration And Invasion ◽  
Mrna Levels ◽  
High Grade ◽  
Protein Levels ◽  
Adult Glioma

Abstract H3.3K27M is a newly identified molecular pathology marker in glioma and is especially correlated with the malignancy of diffuse intrinsic pontine glioma (DIPG). In recent years, accumulating research has revealed that other types of glioma also contain the H3.3K27M mutation. However, the role of H3.3K27M in high-grade adult glioma, which is the most malignant glioma, has not been investigated. In this study, we focused on exploring the expression and function of H3.3K27M in high-grade adult glioma patients. We found that H3.3K27M is partly highly expressed in high-grade glioma tissues. Then, we introduced H3.3K27M into H3.3 wild-type glioma cells, U87 cells and LN229 cells. We found that H3.3K27M did not regulate the growth of glioma in vitro and in vivo; however, the survival of mice with transplanted tumors was significantly reduced. Further investigation revealed that H3.3K27M expression mainly promoted the migration and invasion of glioma cells. Moreover, we certified that H3.3K27M overexpression enhanced the protein levels of ꞵ-catenin and p-ꞵ-catenin, the protein and mRNA levels of ubiquitin-specific protease 1 (USP1), and the protein level of enhancer of zeste homolog 2 (EZH2). Importantly, the ꞵ-catenin inhibitor XAV-939 significantly attenuated the upregulation of the aforementioned proteins. Overall, the H3.3K27M mutation is present in a certain proportion of high-grade glioma patients and facilitates a poor prognosis by promoting the metastasis of glioma by regulating the ꞵ-catenin/USP1/EZH2 pathway.

scholarly journals Sox2-dependent maintenance of mouse oligodendroglioma involves the Sox2-mediated downregulation of Cdkn2b, Ebf1, Zfp423 and Hey2

2020 ◽  
Author(s):  
Cristiana Barone ◽  
Mariachiara Buccarelli ◽  
Francesco Alessandrini ◽  
Miriam Pagin ◽  
Laura Rigoldi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Transcription Factor ◽  
Cancer Stem Cells ◽  
Regulatory Network ◽  
Glioma Cells ◽  
High Grade Glioma ◽  
High Grade

AbstractCancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain tumors. In gliomas, Sox2 is essential to maintain CSC. In mouse high-grade glioma pHGG, Sox2 deletion causes cell proliferation arrest and inability to reform tumors in vivo; 134 genes are significantly derepressed. To identify genes mediating the effects of Sox2 deletion, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Cdkn2b, Ebf1, Zfp423 and Hey2, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis, or pharmacological inactivation, of each of these genes, individually, showed that their activity is essential for the proliferation arrest caused by Sox2 deletion. These Sox2-inhibited antioncogenes also inhibited clonogenicity in primary human glioblastoma-derived cancer stem-like cell lines. These experiments identify critical anti-oncogenic factors whose inhibition by Sox2 is involved in CSC maintenance, defining new potential therapeutic targets for gliomas.Table of Contents ImageMain PointsSox2 maintains glioma tumorigenicity by repressing the antioncogenic activity of a regulatory network involving the Ebf1, Hey2, Cdkn2b and Zfp423 genes.Mutation of these genes prevents the cell proliferation arrest of Sox2-deleted glioma cells.

scholarly journals HGG-02. NEUROPHYSIOLOGICAL SMALL MOLECULE SCREEN TO TARGET NEURON-GLIOMA INTERACTIONS IN PEDIATRIC HIGH-GRADE GLIOMAS

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i17-i17
Author(s):  
David Rogawski ◽  
Sara Mulinyawe ◽  
Craig Thomas ◽  
Michelle Monje
Keyword(s):  
Ion Channels ◽  
Small Molecule ◽  
Glioma Cells ◽  
High Grade Glioma ◽  
High Grade ◽  
In Vivo Experiments ◽  
Target Neuron ◽  
Glioma Cell Proliferation ◽  
Glioma Growth

Abstract Neurons stimulate glioma growth via synaptic and paracrine signaling mechanisms. We recently demonstrated that neurons form AMPA receptor-dependent synapses with glioma cells, and that neuronal activity also induces potassium-evoked currents that are amplified by gap junctions coupling glioma cells. However, our understanding of the neurotransmitters, receptors, and ion channels participating in neuron-glioma signaling remains incomplete. We have recently developed a high-throughput neuron-glioma co-culture strategy to screen small molecules for agents that may disrupt neuron-glioma signaling. Glioma cell proliferation is increased tenfold when cultured together with neurons; this robust biological effect can be probed in a targeted screen of compounds influencing neurotransmitter receptors and ion channels. The neurophysiological small molecule library used was curated to include approved anti-epileptics, neuroleptics, and antidepressants, as well as a variety of other compounds acting on different neurotransmitter types and ion channels. Hits from the primary screen were run through a counter-screen using glioma cells grown alone without neurons, to identify compounds that specifically affect neuron-glioma interactions. Correlation of the screening results with drug mechanisms of action will allow us to map out the key neurotransmitter pathways regulating glioma growth, which can be further validated using genetic and in vivo experiments. Drugs identified in this glioma neuroscience screen may be readily translated into much-needed therapeutics for children with high-grade glioma.

scholarly journals Gene editing preserves visual function in a mouse model of retinal degeneration

2019 ◽  
Author(s):  
Paola Vagni ◽  
Laura E. Perlini ◽  
Naïg A. L. Chenais ◽  
Tommaso Marchetti ◽  
Martina Parrini ◽  
...  
Keyword(s):  
Mouse Model ◽  
Retinitis Pigmentosa ◽  
Gene Editing ◽  
Point Mutations ◽  
Loss Of Function ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Function Point

AbstractInherited retinal dystrophies are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favourable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. We used a tailored CRISPR/Cas9-based gene editing system to prevent retinal photoreceptor death in the Rd10 mouse model of retinitis pigmentosa. We tested the gene editing toolin vitroand then usedin vivosubretinal electroporation to deliver it to one of the retinas of mouse pups at different stages of photoreceptor differentiation. Three months after gene editing, the treated eye exhibited a higher visual acuity compared to the untreated eye. Moreover, we observed preservation of light-evoked responses both in explanted retinas and in the visual cortex of treated animals. Our study validates a CRISPR/Cas9-based therapy as a valuable new approach for the treatment of retinitis pigmentosa caused by autosomal recessive loss-of-function point mutations.

scholarly journals THADA fusion is a mechanism of IGF2BP3 activation and IGF1R signaling in thyroid cancer

2017 ◽  
Vol 114 (9) ◽  
pp. 2307-2312 ◽  
Author(s):  
Federica Panebianco ◽  
Lindsey M. Kelly ◽  
Pengyuan Liu ◽  
Shan Zhong ◽  
Sanja Dacic ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Cancer Cells ◽  
Point Mutations ◽  
Chimeric Protein ◽  
Driver Mutations ◽  
Chromosome 2 ◽  
Igf1r Signaling ◽  
Genome Analyses

Thyroid cancer development is driven by known point mutations or gene fusions found in ∼90% of cases, whereas driver mutations in the remaining tumors are unknown. The insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) plays an important role in cancer, yet the mechanisms of its activation in cancer cells remain poorly understood. Using whole-transcriptome and whole-genome analyses, we identified a recurrent fusion between the thyroid adenoma-associated (THADA) gene on chromosome 2 and the LOC389473 gene on chromosome 7 located 12 kb upstream of the IGF2BP3 gene. We show that THADA fusion to LOC389473 and other regions in the vicinity does not result in the formation of a chimeric protein but instead leads to strong overexpression of the full-length IGF2BP3 mRNA and protein, increased IGF2 translation and IGF1 receptor (IGF1R) signaling via PI3K and MAPK cascades, and promotion of cell proliferation, invasion, and transformation. THADA fusions and IGF2BP3 overexpression are found in ∼5% of thyroid cancers that lack any other driver mutations. We also find that strong IGF2BP3 overexpression via gene fusion, amplification, or other mechanisms occurs in 5 to 15% of several other cancer types. Finally, we provide in vitro and in vivo evidence that growth of IGF2BP3-driven cells and tumors may be blocked by IGF1R inhibition, raising the possibility that IGF2BP3 overexpression in cancer cells may predict an anti-IGF1R benefit.

Glial cytotoxicity of low doses of cadmium as a model of heavy metal pollution influence on vertebrates

2020 ◽  
Vol 31 (1) ◽  
pp. 3-10
Author(s):  
V. S. Nedzvetsky ◽  
V. Ya. Gasso ◽  
A. M. Hahut ◽  
I. A. Hasso
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Oxidative Damage ◽  
Cadmium Chloride ◽  
Glioma Cells ◽  
Low Doses ◽  
Genotoxic Effects ◽  
Cadmium Ions

Cadmium is a common transition metal that entails an extremely wide range of toxic effects in humans and animals. The cytotoxicity of cadmium ions and its compounds is due to various genotoxic effects, including both DNA damage and chromosomal aberrations. Some bone diseases, kidney and digestive system diseases are determined as pathologies that are closely associated with cadmium intoxication. In addition, cadmium is included in the list of carcinogens because of its ability to initiate the development of tumors of several forms of cancer under conditions of chronic or acute intoxication. Despite many studies of the effects of cadmium in animal models and cohorts of patients, in which cadmium effects has occurred, its molecular mechanisms of action are not fully understood. The genotoxic effects of cadmium and the induction of programmed cell death have attracted the attention of researchers in the last decade. In recent years, the results obtained for in vivo and in vitro experimental models have shown extremely high cytotoxicity of sublethal concentrations of cadmium and its compounds in various tissues. One of the most studied causes of cadmium cytotoxicity is the development of oxidative stress and associated oxidative damage to macromolecules of lipids, proteins and nucleic acids. Brain cells are most sensitive to oxidative damage and can be a critical target of cadmium cytotoxicity. Thus, oxidative damage caused by cadmium can initiate genotoxicity, programmed cell death and inhibit their viability in the human and animal brains. To test our hypothesis, cadmium cytotoxicity was assessed in vivo in U251 glioma cells through viability determinants and markers of oxidative stress and apoptosis. The result of the cell viability analysis showed the dose-dependent action of cadmium chloride in glioma cells, as well as the generation of oxidative stress (p <0.05). Calculated for 48 hours of exposure, the LD50 was 3.1 μg×ml-1. The rates of apoptotic death of glioma cells also progressively increased depending on the dose of cadmium ions. A high correlation between cadmium concentration and apoptotic response (p <0.01) was found for cells exposed to 3–4 μg×ml-1 cadmium chloride. Moreover, a significant correlation was found between oxidative stress (lipid peroxidation) and induction of apoptosis. The results indicate a strong relationship between the generation of oxidative damage by macromolecules and the initiation of programmed cell death in glial cells under conditions of low doses of cadmium chloride. The presented results show that cadmium ions can induce oxidative damage in brain cells and inhibit their viability through the induction of programmed death. Such effects of cadmium intoxication can be considered as a model of the impact of heavy metal pollution on vertebrates.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

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