Beyond the Influence of IDH Mutations: Exploring Epigenetic Vulnerabilities in Chondrosarcoma

Mutations in the isocitrate dehydrogenase (IDH1 or IDH2) genes are common in enchondromas and chondrosarcomas, and lead to elevated levels of the oncometabolite D-2-hydroxyglutarate causing widespread changes in the epigenetic landscape of these tumors. With the use of a DNA methylation array, we explored whether the methylome is altered upon progression from IDH mutant enchondroma towards high-grade chondrosarcoma. High-grade tumors show an overall increase in the number of highly methylated genes, indicating that remodeling of the methylome is associated with tumor progression. Therefore, an epigenetics compound screen was performed in five chondrosarcoma cell lines to therapeutically explore these underlying epigenetic vulnerabilities. Chondrosarcomas demonstrated high sensitivity to histone deacetylase (HDAC) inhibition in both 2D and 3D in vitro models, independent of the IDH mutation status or the chondrosarcoma subtype. siRNA knockdown and RNA expression data showed that chondrosarcomas rely on the expression of multiple HDACs, especially class I subtypes. Furthermore, class I HDAC inhibition sensitized chondrosarcoma to glutaminolysis and Bcl-2 family member inhibitors, suggesting that HDACs define the metabolic state and apoptotic threshold in chondrosarcoma. Taken together, HDAC inhibition may represent a promising targeted therapeutic strategy for chondrosarcoma patients, either as monotherapy or as part of combination treatment regimens.

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MODL-20. A BIOBANK OF ~100 PATIENT-DERIVED MODELS REPRESENTING BIOLOGICAL HETEROGENEITY AND DISTINCT THERAPEUTIC DEPENDENCIES IN PAEDIATRIC HIGH GRADE GLIOMA AND DIPG

Neuro-Oncology ◽

10.1093/neuonc/noaa222.593 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii414-iii415

Author(s):

Diana Carvalho ◽

Alan Mackay ◽

Sara Temelso ◽

Elisa Izquierdo ◽

Elisabet Potente Fernandez ◽

...

Keyword(s):

High Grade Glioma ◽

In Vitro Models ◽

Rapid Screening ◽

Mycn Amplification ◽

High Grade ◽

Methylation Array ◽

Original Tumour ◽

Orthotopic Xenografts

Abstract Paediatric high-grade glioma comprise multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived models represent useful tools for mechanistic and preclinical investigations based upon their retention of key genetic/epigenetic features and their amenability to high-throughput approaches. We have collected ~100 in vitro models representing multiple subtypes (H3.3/H3.2/H3.1K27M, H3.3G34R/V, BRAF, MYCN_amp, NTRK_fusion, hypermutator, others) established under 2D (laminin) and/or 3D (neurosphere) conditions, credentialed by phenotypic (growth, invasion/migration) and molecular (methylation array, DNA sequencing, RNAseq) comparison to the original tumour sample. These were derived from patients at our local hospitals (n=29), as part of national co-clinical trials (n=19), from international collaborating centres (n=11), or shared directly by research groups worldwide (n=45). These have variously been subjected to pharmacological (approved/experimental drug libraries) and/or genetic screening (whole-genome CRISPR) to identify specific biological dependencies. Many have been established as orthotopic xenografts in vivo (PDX), with detailed pathological and radiological correlations with the clinical disease, and with tumorigenic latencies ranging from 48–435 days. This resource has allowed us to identify genotype-specific synthetic lethalities and responses to targeted inhibitors, including olaparib (PARP) with ATRX, nutlin-3 (MDM2) with PPM1D, AZD1775 (WEE1) with TP53, and CYC065 (CDK9) with MYCN-amplification. Combinatorial screening highlighted synergies in ACVR1-mutant DIPG between novel ALK2 inhibitors and ONC201 (DRD2). Rapid screening allows for feedback of drug sensitivities to treating clinicians at relapse, whilst mechanistic underpinning of these interactions and use of the models to identify specific mediators of resistance will allow for rational future trial design.

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Advanced Spheroid, Tumouroid and 3D Bioprinted In-Vitro Models of Adult and Paediatric Glioblastoma

International Journal of Molecular Sciences ◽

10.3390/ijms22062962 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2962

Author(s):

Louise Orcheston-Findlay ◽

Samuel Bax ◽

Robert Utama ◽

Martin Engel ◽

Dinisha Govender ◽

...

Keyword(s):

Life Expectancy ◽

High Throughput Screening ◽

High Grade Glioma ◽

Tumour Microenvironment ◽

In Vitro Models ◽

Treatment Modalities ◽

High Grade ◽

Future Improvement ◽

Complex Models

The life expectancy of patients with high-grade glioma (HGG) has not improved in decades. One of the crucial tools to enable future improvement is advanced models that faithfully recapitulate the tumour microenvironment; they can be used for high-throughput screening that in future may enable accurate personalised drug screens. Currently, advanced models are crucial for identifying and understanding potential new targets, assessing new chemotherapeutic compounds or other treatment modalities. Recently, various methodologies have come into use that have allowed the validation of complex models—namely, spheroids, tumouroids, hydrogel-embedded cultures (matrix-supported) and advanced bioengineered cultures assembled with bioprinting and microfluidics. This review is designed to present the state of advanced models of HGG, whilst focusing as much as is possible on the paediatric form of the disease. The reality remains, however, that paediatric HGG (pHGG) models are years behind those of adult HGG. Our goal is to bring this to light in the hope that pGBM models can be improved upon.

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Abstract B14: Establishment and characterization of in vitro models of alternative lengthening of telomeres (ALT) in pediatric high-grade glioma

10.1158/1538-7445.pedca17-b14 ◽

2018 ◽

Author(s):

Jacqueline A. Brosnan-Cashman ◽

Mindy K. Graham ◽

Anthony J. Rizzo ◽

Kaylar Myers ◽

Rebecca Zhang ◽

...

Keyword(s):

High Grade Glioma ◽

In Vitro Models ◽

High Grade ◽

Alternative Lengthening Of Telomeres ◽

Alternative Lengthening ◽

Pediatric High Grade Glioma

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93 SEX-SPECIFIC DEVELOPMENTAL PROGRAMMING OF THE BOVINE EMBRYO BY COLONY STIMULATING FACTOR 2 (CSF2)

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab93 ◽

2014 ◽

Vol 26 (1) ◽

pp. 160 ◽

Cited By ~ 1

Author(s):

K. B. Dobbs ◽

D. Gagné ◽

E. Fournier ◽

I. Dufort ◽

C. Robert ◽

...

Keyword(s):

Linear Models ◽

Statistical Significance ◽

Developmental Programming ◽

Bovine Embryo ◽

Colony Stimulating Factor ◽

Methylation Array ◽

Food And Agriculture ◽

Dna Methylation Array ◽

Stimulating Factor

Competence of the bovine embryo to establish pregnancy can be enhanced by treatment with colony stimulating factor 2 (CSF2) from Days 5 to 7 post-insemination. The mechanism is unknown but could involve developmental reprogramming. Here, we questioned whether treatment with CSF2 from Days 5 to 7 alters growth, interferon-τ (IFNT) secretion, and the methylome of extra-embryonic membranes (EEM) at Day 15. In vitro-produced embryos were treated with either 0 or 10 ng mL–1 bovine CSF2 from Days 5 to 7. Expanded blastocysts were transferred into synchronized recipients. On Day 15, conceptuses were recovered by flushing the uterus and sex determined by PCR (n = 4 males and 4 females per treatment). Statistical analysis of IFNT and length was performed using analysis of variance with the general linear models procedure of SAS (SAS Institute Inc., Cary, NC, USA). There was a sex × treatment interaction for conceptus length (P < 0.003) and IFNT in uterine flushing (P < 0.05) (as determined by antiviral activity). CSF2 increased length in males (least squares means ± s.e.m.: 93 ± 19 mm v. 20 ± 19 mm) and decreased length in females (22 ± 19 mm v. 103 ± 19). Similarly, CSF2 increased IFNT in males (45 483 ± 22 615 IU mL–1 v. –2536.27 ± 13 385 IU mL–1) and decreased IFNT in females (–14 012 ± 13 642 IU mL–1 v. 35 404 ± 7514.91 IU mL–1). The EmbryoGENE DNA Methylation Array (Edmonton, AB, Canada) was used to assess CSF2 effects at 418 805 positions across the genome in a subset of EEM (n = 2 for vehicle and 4 for CSF2 in males; n = 3 for vehicle and n = 3 for CSF2 in females). CSF2 caused hypermethylation for 9842 probes in males and 6227 probes in females and hypomethylation for 9322 probes in males and 3292 probes in females. An analysis was conducted to evaluate if clusters of differentially-methylated probes were non-randomly distributed spatially in 5 Mb regions of individual chromosomes using a 500-kb moving window. Statistical significance was determined using chi-squared tests for each 500-kb window. Differential methylation was not uniformly distributed but rather there were regions or hyper- and hypomethylation that varied with sex. The most common situation was where a region was differentially methylated in one sex but not the other. In some cases, a region was differentially methylated in opposite directions for males and females. For example, a region of BTA1 between positions 67 Mb–77.5 Mb contained 1632 probes, of which 59 probes were hypo-methylated in males and 35 were hypermethylated in females. In conclusion, changes in developmental programming of the bovine embryo caused by CSF2 occur in a sex-specific manner. This result suggests a possible mechanism by which environmental effects on the female affect male embryos differently than female embryos. Support was provided by U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) grant 2011-67015-30688.

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TMOD-06. CREATION OF PATIENT-DERIVED LOWER GRADE GLIOMA ORGANOID MODELS FOR PERSONALIZED TREATMENT RESPONSE ASSESSMENT

Neuro-Oncology ◽

10.1093/neuonc/noab196.868 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi216-vi217

Author(s):

Kalil Abdullah ◽

Joseph Buehler ◽

Cylaina Bird ◽

MIlan Savani ◽

Alex Sternisha ◽

...

Keyword(s):

Treatment Response ◽

Genetic Alterations ◽

Lower Grade ◽

High Grade ◽

Who Grade ◽

Oncology Research ◽

Genetically Engineered Mice ◽

Lower Grade Glioma ◽

Idh Mutations

Abstract Creating in vitro models of lower grade glioma represents a major challenge in neuro-oncology research. There are few such models that are tractable and widely used, which has hindered understanding of the biology of these tumors. Recently, substantial progress has been made in generating patient-derived in vitro organoid models of high grade glioma, but modeling lower grade disease remains difficult. Based on our experience creating neurosphere cultures of lower grade astrocytomas from genetically engineered mice, we hypothesized that modifying patient-derived organoid generation protocols to incorporate physiological oxygen levels would allow establishment and propagation of lower grade glioma organoids. In this study, we show that this approach supports efficient organoid model generation from primary glioma specimens across all histological subtypes and tumor grades (WHO Grade I-IV, n = 20). These organoid models retain key characteristics of their respective parental tumors, including IDH mutations and other genetic alterations, metabolite profiles, intratumoral heterogeneity, cellular composition, and cytoarchitectural features. Importantly, lower grade glioma organoids can be cultured for months and reanimated after biobanking. Our high success rate ( >90%) in establishing organoid models from primary lower grade glioma tissue samples further highlighted opportunities for treatment response assessments. To perform longitudinal measurements of therapy-induced changes in glioma organoid viability, we designed a novel, non-invasive imaging assay (termed rapid apex imaging) to determine real-time treatment response in low and high grade gliomas. We evaluated longitudinal responses of glioblastoma and IDH1 R132H-positive Grade II astrocytoma organoids to temozolomide and olaparib with and without radiation treatment. We quantified topological changes in organoid structure by building a bioinformatics tool to translate imaging data into a cellularity metric as a biomarker of organoid response. Our work unveils an effective new method to create in vitro, personalized models of lower grade glioma that supports elucidation of treatment sensitivity profiles.

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152 The Tumour Suppressor SOX11 is Associated With Improved Cancer-specific Survival Among High Grade Epithelial Ovarian Cancers and is Silenced in Non-expressing in Vitro Models Through Reversible Promoter Methylation

European Journal of Cancer ◽

10.1016/s0959-8049(12)70852-3 ◽

2012 ◽

Vol 48 ◽

pp. S37

Author(s):

S. Sernbo ◽

E. Gustavsson ◽

D.J. Brennan ◽

W.M. Gallagher ◽

E. Rexhepaj ◽

...

Keyword(s):

Promoter Methylation ◽

In Vitro Models ◽

Tumour Suppressor ◽

High Grade ◽

Cancer Specific Survival ◽

Ovarian Cancers

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Class I-Histone Deacetylase (HDAC) Inhibition is Superior to pan-HDAC Inhibition in Modulating Cisplatin Potency in High Grade Serous Ovarian Cancer Cell Lines

International Journal of Molecular Sciences ◽

10.3390/ijms20123052 ◽

2019 ◽

Vol 20 (12) ◽

pp. 3052 ◽

Cited By ~ 8

Author(s):

Jan J. Bandolik ◽

Alexandra Hamacher ◽

Christian Schrenk ◽

Robin Weishaupt ◽

Matthias U. Kassack

Keyword(s):

Ovarian Cancer ◽

Histone Deacetylase ◽

Cell Lines ◽

Hdac Inhibitors ◽

Class I ◽

High Grade ◽

Serous Ovarian Cancer ◽

Hdac Inhibition ◽

Acquired Drug Resistance ◽

Cisplatin Sensitivity

High grade serous ovarian cancer (HGSOC) is the most common and aggressive ovarian cancer subtype with the worst clinical outcome due to intrinsic or acquired drug resistance. Standard treatment involves platinum compounds. Cancer development and chemoresistance is often associated with an increase in histone deacetylase (HDAC) activity. The purpose of this study was to examine the potential of HDAC inhibitors (HDACi) to increase platinum potency in HGSOC. Four HGSOC cell lines with different cisplatin sensitivity were treated with combinations of cisplatin and entinostat (class I HDACi), panobinostat (pan-HDACi), or nexturastat A (class IIb HDACi), respectively. Inhibition of class I HDACs by entinostat turned out superior in increasing cisplatin potency than pan-HDAC inhibition in cell viability assays (MTT), apoptosis induction (subG1), and caspase 3/7 activation. Entinostat was synergistic with cisplatin in all cell lines in MTT and caspase activation assays. MTT assays gave combination indices (CI values) < 0.9 indicating synergism. The effect of HDAC inhibitors could be attributed to the upregulation of pro-apoptotic genes (CDNK1A, APAF1, PUMA, BAK1) and downregulation of survivin. In conclusion, the combination of entinostat and cisplatin is synergistic in HGSOC and could be an effective strategy for the treatment of aggressive ovarian cancer.

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The need for individual selection of probiotics containing lactobacilli and enterococci to increase the effectiveness of therapy for campylobacteriosis

Experimental and Clinical Gastroenterology ◽

10.31146/1682-8658-ecg-186-2-88-93 ◽

2021 ◽

pp. 88-93

Author(s):

K. D. Ermolenko ◽

N. P. Boldyreva ◽

E. A. Martens ◽

L. I. Zhelezova ◽

S. V. Sidorenko ◽

...

Keyword(s):

Antimicrobial Agents ◽

Specific Activity ◽

High Sensitivity ◽

Saccharomyces Boulardii ◽

Antagonistic Activity ◽

Individual Selection ◽

Treatment Regimens ◽

Campylobacter Spp ◽

Selection Of

The article highlights the problem of improving the rational treatment of campylobacteriosis. Probiotics are present in treatment regimens along with antibiotics, which have the advantage that they do not violate intestinal microbiocenosis and provide the ability to correct dysbiotic conditions. As well as antimicrobial agents, probiotics have different effects on the growth of pathogenic microorganisms. Campylobacter spp. probiotics in the in vitro system. The article studies the anticampylobacter activity of probiotic cultures of Enterococcus faecium L3, Lactobacillus plantarum 8 R-A3, a mixture of Lactobacillus acidophilus and Saccharomyces boulardii by two-layer agar and droplet method. Analysis of the antagonistic activity of chemically synthesized bacteriocins. The high sensitivity of Campylobacter spp was presented. to probiotics having lactobacilli and enterococci, as well as their metabolites (including bacteriocins). The strain-specific activity of probiotics and its dependence on their ability to produce bacteriocins were found. The results and data of other researchers indicate the need for individual selection of probiotics for the treatment of campylobacteriosis, the feasibility of analyzing the bacteriocinogenicity of the strains and testing their effect on the growth of clinical isolates.

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