Expression of the hypoxia-inducible factor (HIF)-1-regulated gene product, vascular endothelial growth factor (VEGF), correlates with tumor vascularity in patients with uveal melanoma (UM). While the relationship between HIF-1 and VEGF in cancer is well-studied, their relative contribution to the angiogenic phenotype in UM has not previously been interrogated. Here we evaluate the contribution of HIF-1, VEGF, and a second HIF-1-regulated gene product, angiopoietin-like 4 (ANGPTL4), to angiogenesis in UM.

We investigated 7 male patients (from 5 different families) presenting with profound lymphopenia, hypogammaglobulinemia, fluctuating monocytopenia and neutropenia, a poor immune response to vaccine antigens, and increased susceptibility to bacterial and varicella zoster virus infections.

Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.

DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

Liquid biopsies come of age offering unexploited potential to monitor and react to tumor evolution. We developed a cost-effective assay to non-invasively determine the immune status of glioblastoma (GBM) patients. Employing newly developed printed peptide microarrays we assessed the B-cell response against tumor-associated antigens (TAAs) in 214 patients. Firstly, sera of long-term (36+ months, LTS, n=10) and short-term (6-10 months, STS, n=14) surviving patients were screened for prognostic antibodies against 1745 13-mer peptides covering known TAAs (TNC, EGFR, GLEA2, PHF3, FABP5, MAGEA3). Next, survival associations were investigated in two retrospective independent multicenter validation sets (n=61, n=129, all IDH1-wildtype). Reliability of measurements was tested using a second array technology (spotted arrays). LTS/STS screening analyses identified 106 differential antibody responses. Evaluating the Top30 peptides in validation set 1 revealed three prognostic peptides. Prediction of TNC peptide VCEDGFTGPDCAE was confirmed in a second set (p=0.043, HR=0.66 [0.44-0.99]) and was unrelated to TNC protein expression. Median signals of printed arrays correlated with pre-synthesized spotted microarrays (p<0.0002, R=0.33). Multiple survival analysis revealed independence of age, gender, KPI and MGMT status. We present a novel peptide microarray immune assay that identified increased anti-TNC VCEDGFTGPDCAE serum antibody titer as a promising non-invasive biomarker for prolonged survival.

Pilocytic astrocytoma (PA) is the most common primary brain tumor in children; various signaling pathways have been implicated in its biology. The Notch signaling pathway has been found to play a role in the development, stem cell biology, and pathogenesis of several cancers, but its role in PA has not been investigated. We studied alterations in Notch signaling components in tumor tissue from 18 patients with PA and 4 with other low-grade astrocytomas to identify much needed therapeutic targets. We found that Notch pathway members were overexpressed at the mRNA (NOTCH1, NOTCH2, HEY1, HEY2) and protein (HES1) levels in PAs at various anatomic sites compared with non-neoplastic brain samples. These changes were not associated with specific BRAF alterations. Inhibiting the Notch pathway in the pediatric low-grade astrocytoma cell lines Res186 and Res259 using either RNA interference or a γ-secretase inhibitor resulted in variable, but significant, reduction in cell growth and migration. This study suggests a potential role for Notch signaling in pediatric low-grade astrocytoma tumorigenesis and that Notch signaling may be a viable pathway therapeutic target.

Medulloblastoma (MB) is the most common malignant brain tumor in children, where one-third of patients succumb to their disease. This SnapShot describes the classification of MB subgroups, historically by histopathology and currently based on genomic information. Genomics-based classification has identified four major subgroups and provides greater opportunity for developing targeted therapies more successful than current conventional therapy.

Little is known about the molecular alterations that drive formation and growth of conjunctival squamous cell carcinoma (cSCC). We therefore sought to identify genetic changes that could be used as diagnostic markers or therapeutic targets.

Diffuse midline glioma (DMG), H3 K27M-mutant, is a new entity in the updated WHO classification grouping together diffuse intrinsic pontine gliomas and infiltrating glial neoplasms of the midline harboring the same canonical mutation at the Lysine 27 of the histones H3 tail.Two hundred and fifteen patients younger than 18 years old with centrally-reviewed pediatric high-grade gliomas (pHGG) were included in this study. Comprehensive transcriptomic (n = 140) and methylation (n = 80) profiling was performed depending on the material available, in order to assess the biological uniqueness of this new entity compared to other midline and hemispheric pHGG.Tumor classification based on gene expression (GE) data highlighted the similarity of K27M DMG independently of their location along the midline. T-distributed Stochastic Neighbor Embedding (tSNE) analysis of methylation profiling confirms the discrimination of DMG from other well defined supratentorial tumor subgroups. Patients with diffuse intrinsic pontine gliomas (DIPG) and thalamic DMG exhibited a similarly poor prognosis (11.1 and 10.8 months median overall survival, respectively). Interestingly, H3.1-K27M and H3.3-K27M primary tumor samples could be distinguished based both on their GE and DNA methylation profiles, suggesting that they might arise from a different precursor or from a different epigenetic reorganization.These differences in DNA methylation profiles were conserved in glioma stem-like cell culture models of DIPG which mimicked their corresponding primary tumor. ChIP-seq profiling of H3K27me3 in these models indicate that H3.3-K27M mutated DIPG stem cells exhibit higher levels of H3K27 trimethylation which are correlated with fewer genes expressed by RNAseq. When considering the global distribution of the H3K27me3 mark, we observed that intergenic regions were more trimethylated in the H3.3-K27M mutated cells compared to the H3.1-K27M mutated ones.H3 K27M-mutant DMG represent a homogenous group of neoplasms compared to other pediatric gliomas that could be further separated based on the type of histone H3 variant mutated and their respective epigenetic landscapes. As these characteristics drive different phenotypes, these findings may have important implication for the design of future trials in these specific types of neoplasms.

Medulloblastoma is the most common malignant tumor that arises from the cerebellum of the central nervous system. Clinically, medulloblastomas are treated by surgery, radiation, and chemotherapy, all of which result in toxicity and morbidity. Recent reports have identified that DDX3, a member of the RNA helicase family, is mutated in medulloblastoma. In this study, we demonstrate the role of DDX3 in driving medulloblastoma. With the use of a small molecule inhibitor of DDX3, RK-33, we could inhibit growth and promote cell death in two medulloblastoma cell lines, DAOY and UW228, with IC50 values of 2.5 μM and 3.5 μM, respectively. Treatment of DAOY and UW228 cells with RK-33 caused a G1 arrest, resulted in reduced TCF reporter activity, and reduced mRNA expression levels of downstream target genes of the WNT pathway, such as Axin2, CCND1, MYC, and Survivin. In addition, treatment of DAOY and UW228 cells with a combination of RK-33 and radiation exhibited a synergistic effect. Importantly, the combination of RK-33 and 5 Gy radiation caused tumor regression in a mouse xenograft model of medulloblastoma. Using immunohistochemistry, we observed DDX3 expression in both pediatric (55%) and adult (66%) medulloblastoma patients. Based on these results, we conclude that RK-33 is a promising radiosensitizing agent that inhibits DDX3 activity and down-regulates WNT/β-catenin signaling and could be used as a frontline therapeutic strategy for DDX3-expressing medulloblastomas in combination with radiation.

Oligodendrogliomas are diffusely infiltrative gliomas defined by IDH-mutation and co-deletion of 1p/19q. They have highly variable clinical courses, with survivals ranging from 6 months to over 20 years, but little is known regarding the pathways involved with their progression or optimal markers for stratifying risk. We utilized machine-learning approaches with genomic data from The Cancer Genome Atlas to objectively identify molecular factors associated with clinical outcomes of oligodendroglioma and extended these findings to study signaling pathways implicated in oncogenesis and clinical endpoints associated with glioma progression. Our multi-faceted computational approach uncovered key genetic alterations associated with disease progression and shorter survival in oligodendroglioma and specifically identified Notch pathway inactivation and PI3K pathway activation as the most strongly associated with MRI and pathology findings of advanced disease and poor clinical outcome. Our findings that Notch pathway inactivation and PI3K pathway activation are associated with advanced disease and survival risk will pave the way for clinically relevant markers of disease progression and therapeutic targets to improve clinical outcomes. Furthermore, our approach demonstrates the strength of machine learning and computational methods for identifying genetic events critical to disease progression in the era of big data and precision medicine.

Gliomas and meningiomas are the most common brain neoplasms affecting both humans and canines, and identifying druggable targets conserved across multiple brain cancer histologies and comparative species could broadly improve treatment outcomes. While satisfactory cure rates for low grade, non-invasive brain cancers are achievable with conventional therapies including surgery and radiation, the management of non-resectable or recurrent brain tumors remains problematic and necessitates the discovery of novel therapies that could be accelerated through a comparative approach, such as the inclusion of pet dogs with naturally-occurring brain cancers. Evidence supports procaspase-3 as a druggable brain cancer target with PAC-1, a pro-apoptotic, small molecule activator of procaspase-3 that crosses the blood-brain barrier. Procaspase-3 is frequently overexpressed in malignantly transformed tissues and provides a preferential target for inducing cancer cell apoptosis. While preliminary evidence supports procaspase-3 as a viable target in preclinical models, with PAC-1 demonstrating activity in rodent models and dogs with spontaneous brain tumors, the broader applicability of procaspase-3 as a target in human brain cancers, as well as the comparability of procaspase-3 expressions between differing species, requires further investigation. As such, a large-scale validation of procaspase-3 as a druggable target was undertaken across 651 human and canine brain tumors. Relative to normal brain tissues, procaspase-3 was overexpressed in histologically diverse cancerous brain tissues, supporting procaspase-3 as a broad and conserved therapeutic target. Additionally, procaspase-3 expressing glioma and meningioma cell lines were sensitive to the apoptotic effects of PAC-1 at biologically relevant exposures achievable in cancer patients. Importantly, the clinical relevance of procaspase-3 as a potential prognostic variable was demonstrated in human astrocytomas of variable histologic grades and associated clinical outcomes, whereby tumoral procaspase-3 expression was negatively correlated with survival; findings which suggest that PAC-1 might provide the greatest benefit for patients with the most guarded prognoses.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Glioblastoma (GBM) is a typically lethal type of brain tumor with a median survival of 15 months postdiagnosis. This negative prognosis prompted the exploration of alternative treatment options. In particular, the reliance of GBM on angiogenesis triggered the development of anti-VEGF (vascular endothelial growth factor) blocking antibodies such as bevacizumab. Although its application in human GBM only increased progression-free periods but did not improve overall survival, physicians and researchers still utilize this treatment option due to the lack of adequate alternatives. In an attempt to improve the efficacy of anti-VEGF treatment, we explored the role of the egfl7 gene in malignant glioma. We found that the encoded extracellular matrix protein epidermal growth factor-like protein 7 (EGFL7) was secreted by glioma blood vessels but not glioma cells themselves, while no major role could be assigned to the parasitic miRNAs miR-126/126*. EGFL7 expression promoted glioma growth in experimental glioma models in vivo and stimulated tumor vascularization. Mechanistically, this was mediated by an upregulation of integrin α5β1 on the cellular surface of endothelial cells, which enhanced fibronectin-induced angiogenic sprouting. Glioma blood vessels that formed in vivo were more mature as determined by pericyte and smooth muscle cell coverage. Furthermore, these vessels were less leaky as measured by magnetic resonance imaging of extravasating contrast agent. EGFL7-inhibition using a specific blocking antibody reduced the vascularization of experimental gliomas and increased the life span of treated animals, in particular in combination with anti-VEGF and the chemotherapeutic agent temozolomide. Data allow for the conclusion that this combinatorial regimen may serve as a novel treatment option for GBM.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that epigenetically silences gene transcription through histone H3 lysine trimethylation (H3K27me3). EZH2 has been implicated in stem cell maintenance and is overexpressed in hematological and solid malignancie`s including malignant glioma. EZH2 is thought to promote tumor progression by silencing tumor suppressor genes. Hence pharmacological disruption of the PRC2 is an attractive therapeutic strategy for cancer treatment. Here we show that EZH2 is expressed in human glioma and correlates with malignancy. Silencing of EZH2 reduced glioma cell proliferation and invasiveness. While we did not observe induction of cell cycle-associated tumor suppressor genes by silencing or pharmacological inhibition of EZH2, microarray analyses demonstrated a strong transcriptional reduction of the AXL receptor kinase. Neither histone nor DNA methylation appeared to be involved in the positive regulation of AXL by EZH2. Silencing AXL mimicked the antiinvasive effects of EZH2 knockdown. Finally, AXL expression is found in human gliomas with high EZH2 expression. Collectively these data suggest that EZH2 drives glioma invasiveness via transcriptional control of AXL independent of histone or DNA methylation.

Somatic mutations in Isocitrate Dehydrogenase 1 (IDH1) are frequent in low grade and progressive gliomas and are characterized by the production of 2-hydroxyglutarate (2-HG) from α-ketoglutarate by the mutant enzyme. 2-HG is an "oncometabolite" that competitively inhibits α-KG dependent dioxygenases resulting in various widespread cellular changes including abnormal hypermethylation of genomic DNA and suppression of cellular differentiation. Despite the growing understanding of IDH mutant gliomas, the development of effective therapies has proved challenging in part due to the scarcity of endogenous mutant in vivo models. Here we report the generation of an endogenous IDH1 anaplastic astrocytoma model which rapidly grows in vivo, produces 2-HG and exhibits DNA hypermethylation. Using this model, we have demonstrated the preclinical efficacy and mechanism of action of the FDA approved demethylating drug 5-azacytidine in vivo. Long term administration of 5-azacytidine resulted in reduction of DNA methylation of promoter loci, induction of glial differentiation, reduction of cell proliferation and a significant reduction in tumor growth. Tumor regression was observed at 14 weeks and subsequently showed no signs of re-growth at 7 weeks despite discontinuation of therapy. These results have implications for clinical trials of demethylating agents for patients with IDH mutated gliomas.

Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in <5 % of cases and showed no association with increased patient age. The prognostic implications of these mutations were highly subgroup-specific. TERT mutations identified a subset with good and poor prognosis in SHH and Group 4 tumors, respectively. Monosomy 6 was mostly restricted to WNT tumors without TERT mutations. Hallmark SHH focal copy number aberrations and chromosome 10q deletion were mutually exclusive with TERT mutations within SHH tumors. TERT promoter mutations are the most common recurrent somatic point mutation in medulloblastoma, and are very highly enriched in adult SHH and WNT tumors. TERT mutations define a subset of SHH medulloblastoma with distinct demographics, cytogenetics, and outcomes.

Mutation is a fundamental process in tumorigenesis. However, the degree to which the rate of somatic mutation varies across the human genome and the mechanistic basis underlying this variation remain to be fully elucidated. Here, we performed a cross-cancer comparison of 402 whole genomes comprising a diverse set of childhood and adult tumors, including both solid and hematopoietic malignancies. Surprisingly, we found that the inactive X chromosome of many female cancer genomes accumulates on average twice and up to four times as many somatic mutations per megabase, as compared to the individual autosomes. Whole-genome sequencing of clonally expanded hematopoietic stem/progenitor cells (HSPCs) from healthy individuals and a premalignant myelodysplastic syndrome (MDS) sample revealed no X chromosome hypermutation. Our data suggest that hypermutation of the inactive X chromosome is an early and frequent feature of tumorigenesis resulting from DNA replication stress in aberrantly proliferating cells.

Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.

The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1α protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels. In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels. Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.