New Brain Tumor Entities Emerge from Molecular Classification of CNS-PNETs.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Resolving medulloblastoma cellular architecture by single-cell genomics.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Targeting fibroblast growth factor receptors to combat aggressive ependymoma.

Ependymomas (EPN) are central nervous system tumors comprising both aggressive and more benign molecular subtypes. However, therapy of the high-risk subtypes posterior fossa group A (PF-A) and supratentorial RELA-fusion positive (ST-RELA) is limited to gross total resection and radiotherapy, as effective systemic treatment concepts are still lacking. We have recently described fibroblast growth factor receptors 1 and 3 (FGFR1/FGFR3) as oncogenic drivers of EPN. However, the underlying molecular mechanisms and their potential as therapeutic targets have not yet been investigated in detail. Making use of transcriptomic data across 467 EPN tissues, we found that FGFR1 and FGFR3 were both widely expressed across all molecular groups. FGFR3 mRNA levels were enriched in ST-RELA showing the highest expression among EPN as well as other brain tumors. We further identified high expression levels of fibroblast growth factor 1 and 2 (FGF1, FGF2) across all EPN subtypes while FGF9 was elevated in ST-EPN. Interrogation of our EPN single-cell RNA-sequencing data revealed that FGFR3 was further enriched in cycling and progenitor-like cell populations. Corroboratively, we found FGFR3 to be predominantly expressed in radial glia cells in both mouse embryonal and human brain datasets. Moreover, we detected alternative splicing of the FGFR1/3-IIIc variant, which is known to enhance ligand affinity and FGFR signaling. Dominant-negative interruption of FGFR1/3 activation in PF-A and ST-RELA cell models demonstrated inhibition of key oncogenic pathways leading to reduced cell growth and stem cell characteristics. To explore the feasibility of therapeutically targeting FGFR, we tested a panel of FGFR inhibitors in 12 patient-derived EPN cell models revealing sensitivity in the low-micromolar to nano-molar range. Finally, we gain the first clinical evidence for the activity of the FGFR inhibitor nintedanib in the treatment of a patient with recurrent ST-RELA. Together, these preclinical and clinical data suggest FGFR inhibition as a novel and feasible approach to combat aggressive EPN.

Gliomasphere marker combinatorics: multidimensional flow cytometry detects CD44+/CD133+/ITGA6+/CD36+ signature.

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.

Podoplanin expression in primary brain tumors induces platelet aggregation and increases risk of venous thromboembolism.

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.

Spheroid glioblastoma culture conditions as antigen source for dendritic cell-based immunotherapy: spheroid proteins are survival-relevant targets but can impair immunogenic interferon γ production.

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.

A whole-genome scan for Artemisinin cytotoxicity reveals a novel therapy for human brain tumors.

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.

Trabectedin has promising antineoplastic activity in high-grade meningioma.

BACKGROUND: Meningiomas are common intracranial tumors arising from the meninges and usually are benign. However, a few meningiomas have aggressive behavior and, for such patients, effective treatment options are needed. Trabectedin is a novel, marine-derived, antineoplastic agent that has been approved and is used routinely as therapy for advanced soft tissue sarcoma and ovarian cancer. METHODS: The authors investigated the in vitro effects of trabectedin alone and in combination with hydroxyurea, cisplatin, and doxorubicin in primary cell cultures of benign (n = 9), atypical (n = 6), and anaplastic (n = 4) meningiomas using chemosensitivity assays (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT]), Western blot analysis, cell cycle analysis, and immunofluorescent staining. RESULTS: Strong antimeningioma activity of trabectedin was observed and was characterized by distinct cell cycle arrest, down-regulation of multiple cyclins, deregulated expression of cell death-regulatory genes, and massive apoptosis induction. Cytotoxic activity was especially intense in higher grade meningiomas with a half-maximal inhibitory concentration <10 nM. Combination with trabectedin synergistically enhanced the antimeningioma activity of hydroxyurea but also enhanced the activity of doxorubicin and cisplatin. On the basis of these findings, trabectedin was given to 1 patient who had heavily pretreated, anaplastic meningioma, and a favorable response was observed with radiologic disease stabilization, marked reductions in brain edema and requirement for corticosteroids, and improvement of clinical symptoms. However, treatment had to be discontinued after 5 cycles because of adverse drug effects. CONCLUSIONS: The current results indicated that trabectedin may represent a promising new therapeutic option for patients with aggressive meningioma and should be evaluated in prospective clinical studies.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Heme Biosynthesis mRNA Expression Signature: Towards a Novel Prognostic Biomarker in Patients with Diffusely Infiltrating Gliomas.

Diffusely infiltrating gliomas are characterized by a variable clinical course, and thus novel prognostic biomarkers are needed. The heme biosynthesis cycle constitutes a fundamental metabolic pathway and might play a crucial role in glioma biology. The aim of this study was thus to investigate the role of the heme biosynthesis mRNA expression signature on prognosis in a large glioma patient cohort. Glioma patients with available sequencing data on heme biosynthesis expression were retrieved from The Cancer Genome Atlas (TCGA). In each patient, the heme biosynthesis mRNA expression signature was calculated and categorized into low, medium, and high expression subgroups. Differences in progression-free and overall survival between these subgroups were investigated including a multivariate analysis correcting for WHO grade, tumor subtype, and patient age and sex. In a total of 693 patients, progression-free and overall survival showed a strictly monotonical decrease with increasing mRNA expression signature subgroups. In detail, median overall survival was 134.2 months in the low, 79.9 months in the intermediate, and 16.5 months in the high mRNA expression signature subgroups, respectively. The impact of mRNA expression signature on progression-free and overall survival was independent of the other analyzed prognostic factors. Our data indicate that the heme biosynthesis mRNA expression signature might serve as an additional novel prognostic marker in patients with diffusely infiltrating gliomas to optimize postoperative management.

Response of experimental malignant melanoma models to the pan-Aurora kinase inhibitor VE-465.

Aurora kinases represent promising novel cancer therapy targets. Genomic analyses of human cutaneous melanoma (CMM) models (N = 51, low passage) by classical and/or array CGH revealed frequent gains at chromosome 20q (65%, amplifications in 45%) repeatedly including the Aurora A gene locus. Accordingly, the majority of CMM cell cultures overexpressed Aurora A when compared to proliferating non-malignant cells. Moreover, CMM cells even when arrested in G1/S cell cycle phase contained readily detectable levels of Aurora A indicating incomplete degradation during mitosis. Already at low concentrations (10-100 nm), long-term (7-10 days) application of the pan-Aurora kinase inhibitor VE-465 completely prevented colony formation in all CMM models tested. In contrast, blockade of cell survival/proliferation and DNA synthesis as well as the induction of apoptosis by VE-465 distinctly differed in short-term experiments (up to 72 h exposure). Both cell cycle arrest and DNA synthesis blockade depended on the level of VE-465-mediated p53/p21 activation while p53/p21 unresponsiveness led to repetitive endoreduplication (>8n DNA content). In contrast, apoptosis induction by VE-465 and Aurora A siRNA did not correlate with p53/p21 responsiveness and DNA synthesis blockade. Moreover, application of the Aurora B-specific inhibitor ZM447439 and siRNA was less efficient to induce CMM cell death proofing that apoptosis induction by VE-465 depended predominantly on Aurora A targeting. In combination experiments with chemotherapeutic agents, VE-465 acted additive to antagonistic when applied concomitantly but in several cases even synergistic when applied consecutively. In summary, we suggest that the Aurora A kinase might represent a promising target of well-designed novel antimelanoma strategies.

TCGA mRNA Expression Analysis of the Heme Biosynthesis Pathway in Diffusely Infiltrating Gliomas: A Comparison of Typically 5-ALA Fluorescent and Non-Fluorescent Gliomas.

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.

Combined proteomics/miRNomics of dendritic cell immunotherapy-treated glioblastoma patients as a screening for survival-associated factors.

Glioblastoma is the most prevalent and aggressive brain cancer. With a median overall survival of ~15-20 months under standard therapy, novel treatment approaches are desperately needed. A recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cell (DC) vaccination, however, failed to prolong survival. Here, we investigated tumor tissue from trial patients to explore glioblastoma survival-related factors. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n = 36) with microRNA sequencing plus RT-qPCR (n = 38). Protein quantification identified, e.g., huntingtin interacting protein 1 (HIP1), retinol-binding protein 1 (RBP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as factor candidates correlated with a dismal prognosis. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with favorable tissue characteristics as they were enriched in patients with a comparably longer survival. To illustrate the utility of integrated miRNomics and proteomics findings, focal adhesion was studied further as one example for a pathway of potential general interest. Taken together, we here mapped possible drivers of glioblastoma outcome under immunotherapy in one of the largest DC vaccination tissue analysis cohorts so far-demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy-potentially building on insights generated here.

Single-Cell RNA-Seq Reveals Cellular Hierarchies and Impaired Developmental Trajectories in Pediatric Ependymoma.

Ependymoma is a heterogeneous entity of central nervous system tumors with well-established molecular groups. Here, we apply single-cell RNA sequencing to analyze ependymomas across molecular groups and anatomic locations to investigate their intratumoral heterogeneity and developmental origins. Ependymomas are composed of a cellular hierarchy initiating from undifferentiated populations, which undergo impaired differentiation toward three lineages of neuronal-glial fate specification. While prognostically favorable groups of ependymoma predominantly harbor differentiated cells, aggressive groups are enriched for undifferentiated cell populations. The delineated transcriptomic signatures correlate with patient survival and define molecular dependencies for targeted treatment approaches. Taken together, our analyses reveal a developmental hierarchy underlying ependymomas relevant to biological and clinical behavior.

TERT expression is susceptible to BRAF and ETS-factor inhibition in BRAFV600E/TERT promoter double-mutated glioma.

The BRAF gene and the TERT promoter are among the most frequently altered genomic loci in low-grade (LGG) and high-grade-glioma (HGG), respectively. The coexistence of BRAF and TERT promoter aberrations characterizes a subset of aggressive glioma. Therefore, we investigated interactions between those alterations in malignant glioma. We analyzed co-occurrence of BRAFV600E and TERT promoter mutations in our clinical data (n = 8) in addition to published datasets (n = 103) and established a BRAFV600E-positive glioma cell panel (n = 9) for in vitro analyses. We investigated altered gene expression, signaling events and TERT promoter activity upon BRAF- and E-twenty-six (ETS)-factor inhibition by qRT-PCR, chromatin immunoprecipitation (ChIP), Western blots and luciferase reporter assays. TERT promoter mutations were significantly enriched in BRAFV600E-mutated HGG as compared to BRAFV600E-mutated LGG. In vitro, BRAFV600E/TERT promoter double-mutant glioma cells showed exceptional sensitivity towards BRAF-targeting agents. Remarkably, BRAF-inhibition attenuated TERT expression and TERT promoter activity exclusively in double-mutant models, while TERT expression was undetectable in BRAFV600E-only cells. Various ETS-factors were broadly expressed, however, only ETS1 expression and phosphorylation were consistently downregulated following BRAF-inhibition. Knock-down experiments and ChIP corroborated the notion of a functional role for ETS1 and, accordingly, all double-mutant tumor cells were highly sensitive towards the ETS-factor inhibitor YK-4-279. In conclusion, our data suggest that concomitant BRAFV600E and TERT promoter mutations synergistically support cancer cell proliferation and immortalization. ETS1 links these two driver alterations functionally and may represent a promising therapeutic target in this aggressive glioma subgroup.

TERT promoter mutations are associated with poor prognosis and cell immortalization in meningioma.

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.

CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model.

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.

Telomerase activation in posterior fossa group A ependymomas is associated with dismal prognosis and chromosome 1q gain.

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.

Another step toward DNA selective targeting: Ni(II) and Cu(II) complexes of a Schiff base ligand able to bind gene promoter G-quadruplexes.

DNA G-rich sequences are able to form four-stranded structures organized in stacked guanine tetrads. These structures, called G-quadruplexes, were found to have an important role in the regulation of oncogenes expression and became, for such a reason, appealing targets for anticancer drugs. Aiming at finding selective G-quadruplex binders, we have designed, synthesized and characterized a new water soluble Salen-like Schiff base ligand and its Ni(II) and Cu(II) metal complexes. UV-Vis, circular dichroism and FRET measurements indicated that the nickel complex can stabilize oncogene promoter G-quadruplexes with high selectivity, presenting no interactions with duplex DNA at all. The same compound exhibited dose-dependent cytotoxic activity in MCF-7 breast cancer cells when combined with lipofectamine as lipophilic carrier.

Acquired nintedanib resistance in FGFR1-driven small cell lung cancer: role of endothelin-A receptor-activated ABCB1 expression.

Genomically amplified fibroblast growth factor receptor 1 (FGFR1) is an oncogenic driver in defined lung cancer subgroups and predicts sensibility against FGFR1 inhibitors in this patient cohort. The FGFR inhibitor nintedanib has recently been approved for treatment of lung adenocarcinoma and is currently evaluated for small cell lung cancer (SCLC). However, tumor recurrence due to development of nintedanib resistance might occur. Hence, we aimed at characterizing the molecular mechanisms underlying acquired nintedanib resistance in FGFR1-driven lung cancer. Chronic nintedanib exposure of the FGFR1-driven SCLC cell line DMS114 (DMS114/NIN) but not of two NSCLC cell lines induced massive overexpression of the multidrug-resistance transporter ABCB1. Indeed, we proved nintedanib to be both substrate and modulator of ABCB1-mediated efflux. Importantly, the oncogenic FGFR1 signaling axis remained active in DMS114/NIN cells while bioinformatic analyses suggested hyperactivation of the endothelin-A receptor (ETAR) signaling axis. Indeed, ETAR inhibition resensitized DMS114/NIN cells against nintedanib by downregulation of ABCB1 expression. PKC and downstream NFκB were identified as major downstream players in ETAR-mediated ABCB1 hyperactivation. Summarizing, ABCB1 needs to be considered as a factor underlying nintedanib resistance. Combination approaches with ETAR antagonists or switching to non-ABCB1 substrate FGFR inhibitors represent innovative strategies to manage nintedanib resistance in lung cancer.