Contamination of tissue samples with blood is hardly avoidable. The possibilities for replicate sampling and tissue washing are limited when working with resected specimens of human origin. However, its impact on metabolic profiling has rarely been addressed. In this study, we performed elemental analysis and metabolic profiling focusing on 108 hydrophilic target metabolites in 61 meningioma tissue samples obtained from a non-stratified cohort of 36 patients during neurosurgery. We used iron as an easily accessible proxy for blood. The samples were frequently and non-homogenously contaminated with blood, with iron concentrations ranging from 5 to > 160 µg/g fresh weight. Ten metabolites were significantly altered with their concentrations more than halved or more than doubled when comparing samples with low and high degrees of blood contamination. By contrast, blood contamination did not dominate the apparent metabolic heterogeneity between the samples during PCA analysis based on the metabolites targeted in this study and in the context of a heterogeneous patient cohort and non-standardized sampling. However, studies targeting the reported vulnerable metabolites should critically reflect blood contamination as a biasing factor. Moreover, we report estimated concentrations of 74 hydrophilic metabolites to give tissue-specific orientation for future quantification studies.

Pediatric high-grade gliomas (pHGGs) are among the most lethal childhood tumors. While therapeutic approaches were largely adapted from adult treatment regime, significant biological differences between pediatric and adult gliomas exist, which influence the immune microenvironment and may contribute to the limited response to current pHGG treatment strategies. We provide a comprehensive transcriptomic analysis of the pHGG immune landscape using single-cell RNA sequencing and spatial transcriptomics. We analyze matched malignant, myeloid, and T cells from patients with pediatric diffuse high-grade glioma (HGG) or high-grade ependymoma, examining immune microenvironment distinctions after chemo-/radiotherapy, immune checkpoint inhibition treatment, and by age. Our analysis reveals differences in the proportions of pediatric myeloid subpopulations compared to adult counterparts. Additionally, we observe significant shifts toward immune-suppressive environments following cancer therapy. Our findings offer valuable insights into potential immunotherapy targets and serve as a robust resource for understanding immune microenvironmental variations across HGG age groups and treatment regimens.

BACKGROUND:We introduce TRAM, a triple acquisition strategy on a high-speed quadrupole time-of-flight mass spectrometer for merging non-targeted and targeted metabolomics into one run. TRAM stands for "quasi-simultaneous" acquisition of (1) a full scan MS1, (2) top 30 data-dependent MS2 (DDA), and (3) targeted scheduled MS2 for multiple reaction monitoring (MRM) within measurement cycles of ∼1 s. TRAM combines the selectivity and sensitivity of state-of-the-art targeted MRM-based methods with the full scope of non-targeted analysis enabled by high-resolution mass spectrometry. RESULTS:In this work, we deploy a workflow based on hydrophilic interaction liquid chromatography (HILIC). For a broad panel of metabolites, we provide chromatographic retention times, and optimized conditions as a basis for targeted MRM experiments, listing accurate masses and sum formulas for fragment ions (including fully 13C labeled analogs). Validation experiments showed that TRAM offered (1) linear working ranges and limits of quantification comparable to MRM-only methods, (2) enabled accurate quantification in SRM 1950 human plasma reference material, and (3) was equivalent to DDA-only approaches in non-targeted metabolomics. Metabolomics in human cerebrospinal fluid showcased the power of the strategy, emphasizing the need for high coverage/high throughput metabolomics in clinical studies. SIGNIFICANCE:Acquiring up to 30 data-dependent spectra per MS cycle while still offering gold standard absolute quantification down to low nanomolar concentrations, TRAM allows in-depth profiling and reduces required sample volume, time, cost, and environmental impact.

Abstract Embryonal tumors with multilayered rosettes (ETMR) are malignant brain tumors that occur predominantly in infants and young children. Most patients die within two years of diagnosis, and more effective, targeted therapies are urgently needed. To better characterize the oncogenic mechanisms of key driver alterations and to identify novel therapeutic targets, we set out to study the cellular heterogeneity of ETMR using single-cell RNA sequencing. Analyses conducted on >4,000 high-quality cells collected from eleven primary and relapse specimens revealed a common cellular hierarchy across all tumors: A highly proliferative neural stem cell-like population (SOX2+) that gives rise to intermediate progenitors (NEUROD1/NEUROG1+) and more differentiated neuron-like cells (STMN2/4+). These malignant cell populations closely overlap with histological patterns of ETMRs, as confirmed by multiplexed immunofluorescence microscopy on patients’ tumors. Comparison to single-cell datasets from human fetuses indicated high resemblance to normal cortical neurogenesis but also revealed key tumor-specific differences. These include expression of the chromosome 19 miRNA cluster (C19MC, the presumed driver in ~90% of ETMRs), which was restricted to the malignant stem cell-like population. Investigating oncogenic mechanisms of C19MC (comprising 46 miRNA genes) through transcriptome-wide RNA immunoprecipitation analysis, we identified extensive target gene regulation for most C19MC members, including distinct regulators of cell cycle, pluripotency, and neuronal differentiation. Silencing of C19MC families with antisense oligonucleotides resulted in pronounced reduction of ETMR cell line growth, indicating potential avenues for therapeutic targeting in the future. To identify more immediately actionable targets, we investigated inter-cellular signaling between malignant cell populations of ETMRs. Interestingly, we identified marked FGFR and NOTCH receptor-ligand interactions common to all tumors. An in vitro screen of experimental and approved small molecule inhibitors designed to target these interactions nominated several promising candidates for clinical evaluation. Our unpublished results provide much needed insight into targeting ETMR cellular states using multiple modes of action.

Abstract Forward genetic screens have revolutionized the field of target discovery, target deconvolution, and target validation, particularly in the cancer field. However, most approaches are limited to the interrogation of non-essential factors and identify gene networks rather than direct target structures. Inspired from work in the nematode C. elegans, we perform forward genetic screens based on chemical mutagenesis. This approach randomly introduces single nucleotide variants into the entire genome resulting in cell populations which carry substitutions in statistically more than 90% of all amino acids. Challenging such mutagenized cell populations with a drug candidate of interest, followed by next generation sequencing and analysis, reveals drug resistance mechanism, direct drug target interactions, and potential concealed efficacies. The alteration of single amino acids thereby allows interrogation of non-essential as well as most essential factors and increases screening resolution to the amino acid level. From such screens we inferred and tested a novel treatment option for glioblastoma multiforme. The natural compound Artemisinin and its derivative Artesunate are widely used anti-malarial drugs. Based on theircytotoxic activity, they are also tested as anti-cancer therapies, while the definite mechanism of action and critical host cell targets have remained largely elusive. Using forward genetic screening approaches, we demonstrate that porphyrin biosynthesis governs Artemisinin’s cytotoxicity. Genetic or pharmacological modulation of porphyrin production is sufficient to alter Artemisinin cytotoxicity in multiple eukaryotic cells, including human cancer cells. Combining Artesunate with 5-ALA treatment we translated the screening results to clinically relevant model systems of brain tumor development, such as glioblastomas in engineered cerebral organoids, patient-derived brain tumor spheroids, and orthotopic xenograft models. 5-ALA is a clinically approved photodynamic porphyrin enhancer and surgical fluorescence marker, which specifically marks tumorigenic brain areas. With this additional level of selectivity, we demonstrate a strong antineoplastic effect of the 5-ALA-Artesunatecombination in all tested model systems. These findings extend to the triple combination with temozolomide and urgently await further testing in clinical setups. Citation Format: Michael Orthofer, Jasmin Taubenschmid-Stowers, Marianna Rozsova, Anna Laemmerer, Daniela Loetsch, Johannes Gojo, Andreas Peyrl, Walter Berger, Ullrich Elling, Moritz Horn, Josef M. Penninger. Unprecedented forward genetic screens reveal mechanisms of action, drug resistances, and hidden efficacies at amino acid resolution: combination of 5-ALA and Artesunate as a treatment option for glioblastoma multiforme. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3951.

BRAFV600E mutations occur in 10-15% and homozygous CDKN2A deletions in approximately 40% of adolescent and pediatric high-grad glioma (HGG) and is associated with significantly decreased overall survival. Moreover, a subset of pHGG is defined by combined BRAFV600E mutation and concomitant CDKN2A homozygous deletion. Standard therapy has remained unchanged and consists of maximal safe resection and focal radiotherapy still resulting in poor overall survival. Therefore, new therapeutic approaches to increase patient survival are of utmost importance. Targeted therapy of BRAFV600E mutant HGG has emerged as effective treatment but remains challenging due to therapy resistance. CDK4/6 inhibitors represent a promising target in CDKN2A altered HGG. However, palbociclib demonstrated only low efficacy in HGG patients. Therefore, combination approaches may be more effective, and we investigated CDK4/6-inhibitors alone and in combination with trametinib in HGG cell models with BRAFV600E mutation and homozygous CDKN2A deletion. Efficacy of CDK4/6-inhibitor monotherapy and combinatorial approach with trametinib was assessed by short- and long-term viability assays in four patient-derived HGG cell models with homozygous CDKN2A deletion and BRAFV600E mutation. Furthermore, effects of CDK4/6 inhibitor therapy alone or in combination with trametinib on downstream signaling pathways (MAPK, PI3K) were analyzed with Western blots. Abemaciclib showed the highest activity in all cell models with IC50-values ranging from 0,5 – 2µM. Combined treatment approaches with trametinib showed synergistic effects across all cell models. Long-term viability assays revealed distinct sensitivity in the nanomolar range for CDK4/6-inhibitors and combined treatment with trametinib in all cell models. Exposure to abemaciclib and trametinib significantly decreased pRB, pERK, pS6, and pAKT protein expression levels when compared to monotherapy alone. Summarizing, combined treatment with CDK4/6-inhibitors and trametinib showed promising therapeutic effects on HGG models with homozygous CDKN2A deletion and BRAFV600E mutation. Currently, effects on cell cycle distribution and downstream molecular mechanisms are investigated to identify potential predictive biomarkers.

The natural compound Artemisinin is the most widely used antimalarial drug worldwide. Based on its cytotoxicity, it is also used for anticancer therapy. Artemisinin and its derivates are endoperoxides that damage proteins in eukaryotic cells; their definite mechanism of action and host cell targets, however, have remained largely elusive. Using yeast and haploid stem cell screening, we demonstrate that a single cellular pathway, namely porphyrin (heme) biosynthesis, is required for the cytotoxicity of Artemisinins. Genetic or pharmacological modulation of porphyrin production is sufficient to alter its cytotoxicity in eukaryotic cells. Using multiple model systems of human brain tumor development, such as cerebral glioblastoma organoids, and patient-derived tumor spheroids, we sensitize cancer cells to dihydroartemisinin using the clinically approved porphyrin enhancer and surgical fluorescence marker 5-aminolevulinic acid, 5-ALA. A combination treatment of Artemisinins and 5-ALA markedly and specifically killed brain tumor cells in all model systems tested, including orthotopic patient-derived xenografts in vivo. These data uncover the critical molecular pathway for Artemisinin cytotoxicity and a sensitization strategy to treat different brain tumors, including drug-resistant human glioblastomas.

Brain and spinal cord tumors are the second most common group of cancers in children. For children who experience relapses of their tumors, usually after very intensive first-line therapy, curative treatment options are scarce. Thus, the need for predictive preclinical platforms explicitly for pediatric brain tumors is an urgent need. In the framework of the ITCC-P4 public-private partnership, we thus far fully established 23 pediatric brain tumor-derived PDX models. A selection of four high grade glioma, one medulloblastoma and one ependymoma models were used to validate a protocol for fluorescence-based optical imaging. Tumor growth characteristics, latency and histopathology were evaluated for the un-transduced and iRFP713-transduced models side by side. In case of iRFP713-transduced PDX models, tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany). All animals were examined for neurological symptoms daily and body weight was examined twice a week. The latency of the tumor models ranged from 29 days to 180 days. The take rate was 100% across all the models with n=12 NSG mice per setting. The transduction did not influence the take rate, but 30 - 40% more donor material was needed due to viability loss during the overnight transduction. It was not possible to determine the transduction efficiency for iRFP713 in the overnight culture as the signal is getting upregulated only 48h - 72h post transduction, e.g., when the cells are already implanted. The signal was stable for up to 180 days in the slowest tumor model ependymoma HN0579. The fastest model, high grade glioma HG0068, reached termination criteria within 24 days. Histopathological examination was strictly correlated with the tumor load determined by optical imaging in situ and ex vivo (organ imaging). The histopathological investigation of the mouse brains displayed no differences in tumor localization, size, and invasiveness between the transduced and the un-transduced lines. The proliferation rate determined by Ki-67 staining was not influenced by the modification of the cells. Further molecular and phenotypic characterization of the transduced vs the un-transduced PDX will increase the utility of this platform for the development of new drugs and the identification of innovative drug targets. Citation Format: Eva Oswald, Kanstantsin Lashuk, Johannes Gojo, Dorothee Lenhard, Norman Mack, Sonja Krausert, Daniela Lötsch, David Jones, Marcel Kool, Till Milde, Walter Berger, Stefan M. Pfister, Julia Schüler. Establishment and characterization of pediatric brain tumor models in an orthotopic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1673.

Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/single nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets such as IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

5-Aminolevulinic acid (5-ALA) is a fluorescent dye that after metabolization to Protoporphyrin IX (PpIX) by the heme biosynthesis pathway typically leads to visible fluorescence in WHO grade IV but not grade II gliomas. The exact mechanism for high PpIX levels in WHO grade IV gliomas and low PpIX levels in WHO grade II gliomas is not fully clarified. To detect relevant changes in mRNA expression, we performed an in-silico analysis of WHO grade II and IV glioma sequencing datasets provided by The Cancer Genome Atlas (TCGA) to investigate mRNA expression levels of relevant heme biosynthesis genes: Solute Carrier Family 15 Member 1 and 2 (SLC15A1 and SLC15A2), Aminolevulinate-Dehydratase (ALAD), Hydroxymethylbilane-Synthase (HMBS), Uroporphyrinogen-III-Synthase (UROS), Uroporphyrinogen-Decarboxylase (UROD), Coproporphyrinogen-Oxidase (CPOX), Protoporphyrinogen-Oxidase (PPOX), ATP-binding Cassette Subfamily B Member 6 (ABCB6)/G Member 2 (ABCG2) and Ferrochelatase (FECH). Altogether, 258 WHO grade II and 166 WHO grade IV samples were investigated. The mRNA expression levels showed significant differences in 8 of 11 examined genes between WHO grade II and IV gliomas. Significant differences in mRNA expression included increases of HMBS, UROD, FECH and PPOX as well as decreases of SLC15A2, ALAD, UROS and ABCB6 in WHO IV gliomas. Since the majority of changes was found in directions that might actually impair PpIX accumulation in WHO grade IV gliomas, additional studies are needed to analyze the corresponding factors of the heme biosynthesis also on protein level.

Abstract Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets including IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

Malignant brain tumors are the most common cause of solid cancer death in children. Innovative therapies are vital to improve treatment outcomes, but must be developed to enable trafficking across the blood brain barrier (BBB). For this advent, animal models provide important information prior to clinical studies. Among the different in vivo models orthotopic patient-derived xenograft (PDX) models represent the diversity seen in patient tumors and hence replicate response rates in the clinical trials better as compared to other more simplistic models. Especially in the brain tumor field, imaging has a central role in clinical diagnosis and as a prognostic factor to monitor therapy response. It enables longitudinal patient monitoring in a fully translational manner. Magnetic resonance imaging (MRI) and positron emission tomography (PET) are widely used for clinical diagnosis and disease follow up. Choosing the most suitable imaging application depends of the target of interest or mechanism of action. MRI offers unprecedented soft tissue contrast, high spatial resolution and non-invasive nature renders MRI in rodents a perfect tool for preclinical work in oncological applications. In case of orthotopic brain tumor models, MRI offers the state-of-the-art quantitative volumetric tumor size analysis over disease progression. PET is an excellent tool to study tumor proliferation, metabolism, metastasis as well biodistribution of novel antibodies. The purpose of this work was to analyze volumetric, metabolic and functional changes in orthotopic PDX brain tumor model using MRI, MRS and PET imaging. During the course of the experiment, volume, perfusion within the tumor as well proliferation and metastasis were monitored. Perfusion measurement indicates angiogenesis in tumor, one hallmark of most malignant gliomas. Also, alterations in glucose and amino acid metabolism between tumor and healthy tissue has been previously identified. By direct comparison of the imaging data derived from the preclinical mouse model with similar data-sets from the donor patient the translational value of the model as well as the read-out system will be achieved. As a conclusion, translational in vivo imaging techniques were applied to study orthotopic tumor progression. These readouts provide a powerful and translational research tool together with oncological disease animal models allowing comprehensive evaluation of disease progression and treatment interventions for in vivo studies. Citation Format: Tuulia Huhtala, Julia Schueler, Jussi Rytkönen, Daniela Lötsch, Dorothee Lenhard, Artem Shatillo, Kimmo Lehtimäki, Pekka Poutiainen, Diana Miszczuk, Johannes Gojol, Walter Berger. Translational imaging findings in a pediatric patient-derived orthotopic xenograft brain tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1959.

BACKGROUND:Glioblastoma is the most aggressive type of brain cancer. Dendritic cell (DC)-based immunotherapy against glioblastoma depends on the effectiveness of loaded antigens. Sphere-inducing culture conditions are being studied by many as a potential antigen source. Here, we investigated two different in vitro conditions (spheroid culture versus adherent culture) in relation to DC immunotherapy: (1) We studied the specific spheroid-culture proteome and assessed the clinical importance of spheroid proteins. (2) We evaluated the immunogenicity of spheroid lysate - both compared to adherent conditions. METHODS:We used seven spheroid culture systems, three of them patient-derived. Stemness-related markers were studied in those three via immunofluorescence. Spheroid-specific protein expression was measured via quantitative proteomics. The Cancer Genome Atlas (TCGA) survival data was used to investigate the clinical impact of spheroid proteins. Immunogenicity of spheroid versus adherent cell lysate was explored in autologous ELISPOT systems (DCs and T cells from the three patients). RESULTS:(1) The differential proteome of spheroid versus adherent glioblastoma culture conditions could successfully be established. The top 10 identified spheroid-specific proteins were associated with significantly decreased overall survival (TCGA MIT/Harvard cohort; n = 350, P = 0.014). (2) In exploratory experiments, immunogenicity of spheroid lysate vis-á-vis interferon (IFN)γ production was lower than that of adherent cell lysate (IFNγ ELISPOT; P = 0.034). CONCLUSIONS:Spheroid culture proteins seem to represent survival-relevant targets, supporting the use of spheroid culture conditions as an antigen source for DC immunotherapy. However, immunogenicity enhancement should be considered for future research. Transferability of our findings in terms of clinical impact and regarding different spheroid-generation techniques needs further validation.

Glioblastoma is the most dangerous brain cancer. One reason for glioblastoma's aggressiveness are glioblastoma stem‐like cells. To target them, a number of markers have been proposed (CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6). A comprehensive study of co‐expression patterns of them has, however, not been performed so far. Here, we mapped the multidimensional co‐expression profile of these stemness‐associated molecules. Gliomaspheres – an established model of glioblastoma stem‐like cells – were used. Seven different gliomasphere systems were subjected to multicolor flow cytometry measuring the nine markers CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6 all simultaneously based on a novel 9‐marker multicolor panel developed for this study. The viSNE dimensionality reduction algorithm was applied for analysis. All gliomaspheres were found to express at least five different glioblastoma stem‐like cell markers. Multi‐dimensional analysis showed that all studied gliomaspheres consistently harbored a cell population positive for the molecular signature CD44+/CD133+/ITGA6+/CD36+. Glioblastoma patients with an enrichment of this combination had a significantly worse survival outcome when analyzing the two largest available The Cancer Genome Atlas datasets (MIT/Harvard Affymetrix: P = 0.0015, University of North Carolina Agilent: P = 0.0322). In sum, we detected a previously unknown marker combination – demonstrating feasibility, usefulness, and importance of high‐dimensional gliomasphere marker combinatorics.

Central nervous system high-grade neuroepithelial tumor with BCL6-corepressor alteration (CNS HGNET-BCOR) is a recently discovered molecular brain tumor entity characterized by genomic alterations of BCOR, a central component of a non-canonical polycomb repressive complex. However, the underlying oncogenic mechanisms and their consequences for tumor-specific anti-cancer therapy remain widely enigmatic. We systematically analyzed genomics, transcriptomics, and drug-sensitivity patterns in three tumor-derived models (two cell-lines, one primary cell-culture) from three consecutive intracranial CNS HGNET-BCOR metastases of one patient. All models harbored a unique frameshift mutation within BCOR resulting in a truncated protein lacking functionally important c-terminal protein domains. Re-expression of BCOR wild-type in our CNS HGNET-BCOR cell-models resulted in decreased cell proliferation and increased apoptosis. Interestingly, genes downregulated upon re-expression of BCOR wild-type were also derepressed in CNS HGNET-BCOR tumor tissues harboring BCOR-alterations different from our case (e.g. internal tandem duplication in BCOR). Via comparison with a chromatin immunoprecipitation DNA-sequencing dataset, we determined that a significant proportion of the corresponding gene promoters are occupied by BCOR in BCOR wild-type cancer cells. An additional drug-screen demonstrated hypersensitivity of CNS HGNET-BCOR cells against histone deacetylase inhibitors and histone methyltransferase inhibitors. Matching of drug sensitivity patterns to upregulated target-genes determined bortezomib, dasatinib, and crizotinib as promising tumor-specific therapeutics against CNS HGNET-BCOR. Taken together, our results suggest that dysfunction of BCOR-mediated gene repression determines the oncogenic behavior and transcriptomic profile of CNS HGNET-BCOR. Moreover, we provide preliminary results for potential pharmacological interventions against this aggressive tumor type.

Background. Meningiomas are mostly benign tumors tending to progress to higher-grade lesions. Mutations in the telomerase reverse transcriptase (TERT) gene promoter are comparably rare in meningioma, but were recently suggested to predict risk of recurrence and progression. Here we have analyzed a cohort of World Health Organization grades I-III meningiomas regarding the impact of TERT promoter mutations on patient prognosis and in vitro cell propagation feasibility. Methods. From 110 meningioma patients, 128 tissue samples were analyzed for the TERT promoter mutations C228T and C250T by direct sequencing. Of the 128 samples, 121 were tested for cell propagation in vitro. Telomerase activity, TERT mRNA expression, and telomere lengths were investigated by telomeric repeat amplification protocol assay, reverse transcription PCR, and quantitative PCR, respectively. Impact of the E-twenty-six (ETS) transcription factor inhibitor YK-4-279 on cell viability and TERT promoter activity was analyzed. Results. TERT promoter mutations were found in 5.5% of all samples analyzed and were associated with a significantly upregulated telomerase activity and TERT mRNA expression (P < 0.0001 both). Regarding telomere lengths, no significant difference between the TERT promoter wild-type and mutated subgroups was detected. Patients with TERT promoter mutated tumors exhibited significantly shorter overall survival (P = 0.0006; 53.8 vs 115.6 mo). The presence of TERT promoter mutations but not telomerase activity or TERT mRNA expression predicted indefinite cell growth in vitro. TERT promoter mutated meningioma cells were hypersensitive against the ETS transcription factor inhibitor YK-4-279, inducing a distinct downregulation of TERT promoter activity. Conclusion. TERT promoter mutations drive meningioma aggressiveness, resulting in reduced patient survival, but might also open novel therapeutic options for progressive disease.

Background. Ependymomas account for up to 10% of childhood CNS tumors and have a high rate of tumor recurrence despite gross total resection. Recently, classification into molecular ependymoma subgroups has been established, but the mechanisms underlying the aggressiveness of certain subtypes remain widely enigmatic. The aim of this study was to dissect the clinical and biological role of telomerase reactivation, a frequent mechanism of cancer cells to evade cellular senescence, in pediatric ependymoma.Methods. We determined telomerase enzymatic activity, hTERT mRNA expression, promoter methylation, and the rs2853669 single nucleotide polymorphism located in the hTERT promoter in a well-characterized cohort of pediatric intracranial ependymomas.Results. In posterior fossa ependymoma group A (PF-EPN-A) tumors, telomerase activity varied and was significantly associated with dismal overall survival, whereas telomerase reactivation was present in all supratentorial RelA fusion-positive (ST-EPN-RELA) ependymomas. In silico analysis of methylation patterns showed that only these two subgroups harbor hypermethylated hTERT promoters suggesting telomerase reactivation via epigenetic mechanisms. Furthermore, chromosome 1q gain, a well-known negative prognostic factor, was strongly associated with telomerase reactivation in PF-EPN-A. Additional in silico analyses of gene expression data confirmed this finding and further showed enrichment of the E-twenty-six factor, Myc, and E2F target genes in 1q gained ependymomas. Additionally, 1q gained tumors showed elevated expression of ETV3, an E-twenty-six factor gene located on chromosome 1q.Conclusion. Taken together we describe a subgroup-specific impact of telomerase reactivation on disease progression in pediatric ependymoma and provide preliminary evidence for the involved molecular mechanisms.

Background Osteosarcoma is the most common primary malignant bone tumor with a 5 year survival rate of up to 70%. However, patients with metastatic disease have still a very poor prognosis. Osteosarcoma metastasis models are essential to develop novel treatment strategies for advanced disease. Methods Based on a serial transplantation approach, we have established a U-2 OS osteosarcoma xenograft model with increased metastatic potential and compared it to other metastatic osteosarcoma models from international sources. Subclones with differing invasive potential were compared for genomic gains and losses as well as gene expression changes by several bioinformatic approaches. Based on the acquired results, the effects of a shRNA-mediated CD44 mRNA knockdown on migration, invasion and chemosensitivity were evaluated. Results The CD44 gene was part of an amplified region at chromosome 11p found in both U-2 OS subclones with enhanced metastatic potential but not in parental U-2 OS cells, corresponding with distinct CD44 overexpression. Accordingly, shRNA-mediated CD44 knockdown significantly attenuated osteosarcoma cell migration, invasion, and viability especially in the metastatic subclones of U-2 OS and Saos-2 cells. Metastatic subclones generally were hypersensitive against the integrin inhibitor cilengitide paralleled by alterations in integrin expression pattern following CD44 knock-down. Additionally, attenuation of CD44 expression sensitized these cell models against osteosarcoma chemotherapy with doxorubicin but not methotrexate and cisplatin. Conclusions The osteosarcoma xenograft models with increased metastatic potential developed in this study can be useful for identification of mechanisms driving metastasis and resistance towards clinically used and novel therapeutic regimens.

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts (P < .001) and higher D-dimer levels (P < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens (P < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P =010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors.

Recent high-throughput analyses have brought forward novel molecular subgroups of ependymoma, one of the most frequent pediatric brain tumors. However, the mechanisms underlying the distinct behaviour of these subtypes are no entirely clear. The ribonucleotide enzyme telomerase (hTERT), which is capable of elongating shortened telomeres, has been associated with an aggressive course of ependymoma. Herein, we evaluated hTERT promoter methylation patterns, promoter mutation and the rs2853699 within the hTERT promoter as potential mechanisms of telomerase re-activation across different ependymoma subgroups. hTERT promoter mutations were not present in all samples analyzed and levels of telomerase activity and hTERT mRNA expression did not differ among rs2853699 alleles (n=29). Interestingly, no homozygous C allele was observed in ST-EPN-RELA tumors, which might be connected to the disruption of a RelA binding site in this genotype. hTERT promoter methylation tested by pyrosequencing in a promoter region upstream of the rs2853699 was significantly elevated in both posterior fossa and supratentorial ependymomas, the latter showing the highest methylation levels (n=68). Furthermore, hTERT promoter-methylation was significantly associated with higher telomerase activity and Ki67 index but not hTERT mRNA expression. For detailed analysis across molecular ependymoma subgroups, methylation patterns of three additional CpG sites within the hTERT promoter were analyzed in an independent Illumina 450K methylation array dataset (GSE65362, n=500). The CpG site located closest to the transcription start site was hypomethylated in all ependymoma subgroups. In contrast, the more distal sites were almost exclusively hypermethylated in PF-EPN-A and ST-EPN-RELA subtypes. Whereas all ST-EPN-RELA tumors showed hypermethylation, the pattern was more heterogeneous within the PF-EPN-A subgroup. Therefore, we tested the impact of hTERT promoter hypermethylation on survival probabilities within the PF-EPN-A subgroup (n=17), but no significant effect could be determined. Taken together, we present hTERT promoter hypermethylation as potential subgroup-specific mechanism for telomerase re-activation in pediatric ependymoma.