BACKGROUND: Circular RNAs (circRNAs) are a novel type of noncoding RNAs and play important roles in tumorigenesis, including gastric cancer (GC). However, the functions of most circRNAs remain poorly understood. In our study, we aimed to investigate the functions of a new circRNA circ-DONSON in GC progression.
METHODS: The expression of circ-DONSON in gastric cancer tissues and adjacent normal tissues was analyzed by bioinformatics method, qRT-PCR, Northern blotting and in situ hybridization (ISH). The effects of circ-DONSON on GC cell proliferation, apoptosis, migration and invasion were measured by using CCK8, colony formation, EdU, immunofluorescence (IF), FACS and Transwell assays. qRT-PCR and Western blotting were utilized to validate how circ-DONSON regulates SOX4 expression. ChIP, DNA fluorescence in situ hybridization (DNA-FISH) and DNA accessibility assays were used to investigate how circ-DONSON regulates SOX4 transcription. The interaction between circ-DONSON and NURF complex was evaluated by mass spectrum, RNA immunoprecipitation (RIP), pulldown and EMSA assays. Xenograft mouse model was used to analyze the effect of circ-DONSON on GC growth in vivo.
RESULTS: Elevated expression of circ-DONSON was observed in GC tissues and positively associated with advanced TNM stage and unfavorable prognosis. Silencing of circ-DONSON significantly suppressed the proliferation, migration and invasion of GC cells while promoting apoptosis. circ-DONSON was localized in the nucleus, recruited the NURF complex to SOX4 promoter and initiated its transcription. Silencing of the NURF complex subunit SNF2L, BPTF or RBBP4 similarly attenuated GC cell growth and increased apoptosis. circ-DONSON knockdown inhibited GC growth in vivo.
CONCLUSION: circ-DONSON promotes GC progression through recruiting the NURF complex to initiate SOX4 expression.

Loss of SMARCB1 is the hallmark genetic event that characterizes rhabdoid tumors in children. Rhabdoid tumors of the brain (ATRT) occur in young children and are particularly challenging with poor long-term survival. SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. Recent studies demonstrate that ATRT consists of 3 genomic subgroups with a subset of poor outcome tumors expressing high BMP and MYC pathway activation. Here we show that MYC occupies distinct promoter loci in ATRT compared to embryonic stem (ES) cells. Furthermore, using human ATRT cell lines, patient-derived cell culture, ex vivo patient-derived tumor, and orthotopic xenograft models, we show that MYC inhibition is a molecular vulnerability in SMARCB1-deleted tumors and that such inhibition effectively suppresses BMP and pluripotency-associated genomic programs, attenuates tumor cell self-renewal, promotes senescence, and inhibits ATRT tumor growth in vivo. Transgenic expression of Omomyc (a bona-fide MYC dominant negative) or chemical inhibition of MYC transcriptomic programs with the BET inhibitor JQ1 phenocopy genetic depletion of MYC, effectively restricting ATRT tumor growth and opening a promising therapeutic avenue for rhabdoid tumors in children.

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

BACKGROUND: A desperate need for novel therapies in pediatric ependymoma (EPN) exists, as chemotherapy remains ineffective and radiotherapy often fails. EPN have significant infiltration of immune cells, which correlates with outcome. Immune checkpoint inhibitors provide an avenue for new treatments. This study characterizes tumor-infiltrating immune cells in EPN and aims at predicting candidates for clinical trials using checkpoint inhibitors targeting PD-L1/PD-1 (programmed death ligand 1/programmed death 1).
METHODS: The transcriptomic profiles of the primary study cohort of EPN and other pediatric brain tumors were interrogated to identify PD-L1 expression levels. Transcriptomic findings were validated using the western blotting, immunohistochemistry and flow cytometry.
RESULTS: We evaluated PD-L1 mRNA expression across four intracranial subtypes of EPN in two independent cohorts and found supratentorial RELA fusion (ST-RELA) tumors to have significantly higher levels. There was a correlation between high gene expression and protein PD-L1 levels in ST-RELA tumors by both the western blot and immunohistochemisty. The investigation of EPN cell populations revealed PD-L1 was expressed on both tumor and myeloid cells in ST-RELA. Other subtypes had little PD-L1 in either tumor or myeloid cell compartments. Lastly, we measured PD-1 levels on tumor-infiltrating T cells and found ST-RELA tumors express PD-1 in both CD4 and CD8 T cells. A functional T-cell exhaustion assay found ST-RELA T cells to be exhausted and unable to secrete IFNγ on stimulation.
CONCLUSIONS: These findings in ST-RELA suggest tumor evasion and immunsuppression due to PD-L1/PD-1-mediated T-cell exhaustion. Trials of checkpoint inhibitors in EPN should be enriched for ST-RELA tumors.

Diffuse intrinsic pontine glioma (DIPG) is an incurable childhood brain tumor. The mechanistic target of rapamycin (MTOR), a key oncogene, functions as two distinct signaling complexes, MTORC1 and MTORC2. We set out to determine the preclinical efficacy and mechanism of action of MTOR inhibitors in DIPG. We evaluated the MTORC1 inhibitor everolimus and the MTORC1/2 inhibitor AZD2014 in three patient-derived DIPG cell lines using cell culture models. We created dose-response curves for both compounds. We measured phenotypic effects on cell self-renewal, apoptosis, cell cycle, differentiation, senescence, and autophagy. We assessed the effects of each compound on the AKT pathway. Finally, we measured the efficacy of AZD2014 in combination with radiation therapy (RT) and a panel of FDA-approved chemotherapy drugs. While everolimus showed minimal antitumor efficacy, AZD2014 revealed IC50 levels of 410-552 nM and IC90 levels of 1.30-8.86 µM in the three cell lines. AZD2014 demonstrated increased inhibition of cell self-renewal compared to everolimus. AZD2014 decreased expression of phospho-AKT, while no such effect was noted with everolimus. Direct AKT inhibition showed similar efficacy to AZD2014, and induction of constitutive AKT activity rescued DIPG cells from the effects of AZD2014. AZD2014 exhibited synergistic relationships with both RT and various chemotherapy agents across classes, including the multikinase inhibitor ponatinib. MTORC1/2 inhibition shows antitumor activity in cell culture models of DIPG due to the effect of MTORC2 inhibition on AKT. This strategy should be further assessed for potential incorporation into combinatorial approaches to the treatment of DIPG.

Ependymoma (EPN) is a common brain tumor of childhood that, despite standard surgery and radiation therapy, has a relapse rate of 50%. Clinical trials have been unsuccessful in improving outcome by addition of chemotherapy, and identification of novel therapeutics has been hampered by a lack of in vitro and in vivo models. We describe 2 unique EPN cell lines (811 and 928) derived from recurrent intracranial metastases. Both cell lines harbor the high-risk chromosome 1q gain (1q+) and a derivative chromosome 6, and both are classified as molecular group A according to transcriptomic analysis. Transcriptional enrichment of extracellular matrix-related genes was a common signature of corresponding primary tumors and cell lines in both monolayer and 3D formats. EPN cell lines, when cultured in 3D format, clustered closer to the primary tumors with better fidelity of EPN-specific transcripts than when grown as a monolayer. Additionally, 3D culture revealed ependymal rosette formation and cilia-related ontologies, similar to in situ tumors. Our data confirm the validity of the 811 and 928 cell lines as representative models of intracranial, posterior fossa 1q+ EPN, which holds potential to advance translational science for patients affected by this tumor.

Pediatric adamantinomatous craniopharyngioma (ACP) is a highly solid and cystic tumor, often causing substantial damage to critical neuroendocrine structures such as the hypothalamus, pituitary gland, and optic apparatus. Paracrine signaling mechanisms driving tumor behavior have been hypothesized, with IL-6R overexpression identified as a potential therapeutic target. To identify potential novel therapies, we characterized inflammatory and immunomodulatory factors in ACP cyst fluid and solid tumor components. Cytometric bead analysis revealed a highly pro-inflammatory cytokine pattern in fluid from ACP compared to fluids from another cystic pediatric brain tumor, pilocytic astrocytoma. Cytokines and chemokines with particularly elevated concentrations in ACPs were IL-6, CXCL1 (GRO), CXCL8 (IL-8) and the immunosuppressive cytokine IL-10. These data were concordant with solid tumor compartment transcriptomic data from a larger cohort of ACPs, other pediatric brain tumors and normal brain. The majority of receptors for these cytokines and chemokines were also over-expressed in ACPs. In addition to IL-10, the established immunosuppressive factor IDO-1 was overexpressed by ACPs at the mRNA and protein levels. These data indicate that ACP cyst fluids and solid tumor components are characterized by an inflammatory cytokine and chemokine expression pattern. Further study regarding selective cytokine blockade may inform novel therapeutic interventions.

Triple negative breast cancer (TNBC) is an aggressive cancer for which prognosis remains poor. Combination therapy is a promising strategy for enhancing treatment efficacy. Blockade of STAT3 signaling may enhance the response of cancer cells to conventional chemotherapeutic agents. Here we used a SHP-1 agonist SC-43 to dephosphorylate STAT3 thereby suppressing oncogenic STAT3 signaling and tested it in combination with docetaxel in TNBC cells. We first analyzed messenger RNA (mRNA) expression of SHP-1 gene (PTPN6) in a public TNBC dataset (TCGA) and found that higher SHP-1 mRNA expression is associated with better overall survival in TNBC patients. Sequential combination of docetaxel and SC-43 in vitro showed enhanced anti-proliferation and apoptosis associated with decreased p-STAT3 and decreased STAT3-downstream effector cyclin D1 in the TNBC cell lines MDA-MB-231, MDA-MB-468, and HCC-1937. Ectopic expression of STAT3 reduced the increased cytotoxicity induced by the combination therapy. In addition, this sequential combination showed enhanced SHP-1 activity compared to SC-43 alone. Furthermore, the combination treatment-induced apoptosis was attenuated by small interfering RNA (siRNA) against SHP-1 or by ectopic expression of SHP-1 mutants that caused SC-43 to lose its SHP-1 agonist capability. Moreover, combination of docetaxel and SC-43 showed enhanced tumor growth inhibition compared to single-agent therapy in mice bearing MDA-MB-231 tumor xenografts. Our results suggest that the novel SHP-1 agonist SC-43 enhanced docetaxel-induced cytotoxicity by SHP-1 dependent STAT3 inhibition in human triple negative breast cancer cells. TNBC patients with high SHP-1 expressions show better survival. Docetaxel combined with SC-43 enhances cell apoptosis and reduces p-STAT3. SHP-1 inhibition reduces the enhanced effect of docetaxel-SC-43 combination. Docetaxel-SC-43 combination suppresses xenograft tumor growth and reduces p-STAT3.
KEY MESSAGES: TNBC patients with high SHP-1 expressions show better survival. Docetaxel combined with SC-43 enhances cell apoptosis and reduces p-STAT3. SHP-1 inhibition reduces the enhanced effect of docetaxel-SC-43 combination. Docetaxel-SC-43 combination suppresses xenograft tumor growth and reduces p-STAT3.

BACKGROUND: Posterior fossa (PF) ependymomas (EPNs) in infants less than 1 year of age (iEPN-PF) have a poorer clinical outcome than EPNs in older children. While radiation therapy is the standard of care for the latter, it is withheld in infants to avoid neurotoxicity to immature brain. It is unknown whether the adverse outcome in iEPN-PFs is due to treatment differences or aggressive biology. We examined this question using molecular profiling.
METHODS: Six anaplastic iEPN-PFs were subjected to transcriptomic analysis and FISH for p16 loss and gains of 1q, and compared with anaplastic PF EPNs from older children. Results were validated by immunohistochemistry (IHC).
RESULTS: All six iEPN-PFs were grouped within EPN PF subgroup A (PFA). E2F targets and G2M checkpoint were identified as the most enriched gene sets in iEPN-PF, which was validated in a larger independent cohort. Accordingly, MIB-1 IHC demonstrated a higher mitotic rate in iEPN-PFs than noninfant anaplastic EPN PFA. Genetic and protein analyses demonstrated that p16 loss and low p16 protein expression is a hallmark of iEPN-PF, and that none harbored 1q gains. Kaplan-Meier analysis confirmed the poorer clinical outcome of the iEPN-PF cohort.
CONCLUSIONS: Biological differences, characterized by loss of p16 expression without gains of 1q in iEPN-PFs, as well as deregulated E2F target gene transcription, are indicative of deregulated p16-CDK4/6-pRB-E2F pathway activity. This may underlie the poor clinical outcome seen in this group of iEPN-PFs, rather than the withholding of radiation therapy. Results suggest a potential actionable therapy for iEPN-PF, namely cyclin-dependent kinase 4/6 (CDK4/6) inhibitors.

Background: Inflammation has been identified as a hallmark of high-risk Group A (GpA) ependymoma (EPN). Chronic interleukin (IL)-6 secretion from GpA tumors drives an immune suppressive phenotype by polarizing infiltrating monocytes. This study determines the mechanism by which IL-6 is dysregulated in GpA EPN.
Methods: Twenty pediatric GpA and 21 pediatric Group B (GpB) EPN had gene set enrichment analysis for MSigDB Hallmark gene sets performed. Protein and RNA from patients and cell lines were used to validate transcriptomic findings. GpA cell lines 811 and 928 were used for in vitro experiments performed in this study.
Results: The nuclear factor-kappaB (NF-κB) pathway is a master regulator of IL-6 and a signaling pathway enriched in GpA compared with GpB EPN. Knockdown of NF-κB led to significant downregulation of IL-6 in 811 and 928. NF-κB activation was independent of tumor necrosis factor alpha (TNF-α) stimulation in both cell lines, suggesting that NF-κB hyperactivation is mediated through an alternative mechanism. Leucine zipper downregulated in cancer 1 (LDOC1) is a known transcriptional repressor of NF-κB. In many cancers, LDOC1 promoter is methylated, which inhibits gene transcription. We found decreased LDOC1 gene expression in GpA compared with GpB EPN, and in other pediatric brain tumors. EPN cells treated with 5AZA-DC, demethylated LDOC1 regulatory regions, upregulated LDOC1 expression, and concomitantly decreased IL-6 secretion. Stable knockdown of LDOC1 in EPN cell lines resulted in a significant increase in gene transcription of v-rel avian reticuloendotheliosis viral oncogene homolog A, which correlated to an increase in NF-κB target genes.
Conclusion: These results suggest that epigenetic silencing of LDOC1 in GpA EPN regulates tumor biology and drives inflammatory immune phenotype.

Escherichia coli is the most common cause of bloodstream infections and community-acquired sepsis. The main aim of this study was to determine virulence characteristics of E. coli isolates from hemocultures of patients with a primary disease of urogenital tract, digestive system, a neoplastic blood disease, or other conditions. Results from a set of 314 E. coli isolates from hemocultures were compared to data from a previously published analysis of 1283 fecal commensal E. coli isolates. Genetic profiling of the 314 E. coli isolates involved determination of phylogenetic group (A, B1, B2, D, C, E, and F), identification of 21 virulence factors, as well as 30 bacteriocin-encoding determinants. Pulsed-field gel electrophoresis was used to analyze clonal character of the hemoculture-derived isolates. The E. coli isolates from hemocultures belonged mainly to phylogenetic groups B2 (59.9%) and D (21.0%), and less frequently to phylogroups A (10.2%) and B1 (5.7%). Commonly detected virulence factors included adhesins (fimA 92.0%, pap 47.1%, and sfa 26.8%), and iron-uptake encoding genes (fyuA 87.9%, fepC 79.6%, aer 70.7%, iucC 68.2%, and ireA 13.7%), followed by colibactin (pks island 31.5%), and cytotoxic necrotizing factor (cnf1 11.1%). A higher frequency of microcin producers (and microcin M determinant) and a lower frequency of colicin Ib and microcin B17 was found in hemoculture-derived isolates compared to commensal fecal isolates. E. coli isolates from hemocultures harbored more virulence genes compared to fecal E. coli isolates. In addition, hemoculture E. coli isolates from patients with primary diagnosis related to urogenital tract were clearly different and more virulence genes were detected in these isolates compared to both fecal isolates and hemoculture-derived isolates from patients with blood and gastrointestinal diseases.

BACKGROUND: UDP-glucuronosyltransferase 2 family, polypeptide B17 (UGT2B17) encodes for an enzyme that modifies carcinogens, C19 steroids, xenobiotics, and anticancer chemotherapeutic agents by glucuronidation. Pediatric cancers are much more sensitive to anticancer agents than adult cancers. The aim of this study was therefore to examine the effects of UGT2B17 deletion polymorphism on prognosis in pediatric cancer.
METHODS: A total of 145 DNA samples were collected from children with malignant disease. UGT2B17 copy number variant was determined on polymerase chain reaction. Survival analysis was carried out to analyze the effects of UGT2B17 deletion on relapse-free rate in lymphoblastic and non-lymphoblastic malignancy.
RESULTS: UGT2B17 was deleted in 64% of children with lymphoblastic malignancy, but in 83% of children with non-lymphoblastic malignancy. Moreover, in non-lymphoblastic malignancy, children without UGT2B17 deletion polymorphism had significantly higher relapse rates than those with the deletion polymorphism (HR, 16.1; 95%CI: 1.67-154; P = 0.016), which remained significant after adjustment for age, sex, underlying disease, advanced stage, and adverse events (HR, 22.4; 95%CI: 1.10-454; P = 0.043). There was a significant interaction between UGT2B17 deletion and non-lymphoblastic malignancy. In the early subgroup, that is, stages 1-3 or standard/intermediate risk, children without UGT2B17 deletion polymorphism had a higher relapse rate than children with more advanced disease (log-rank test: P = 0.0004).
CONCLUSIONS: UGT2B17 deletion polymorphism may improve the relapse-free rate in children with non-lymphoblastic malignancy.

Medulloblastoma is the most common type of malignant brain tumor that affects children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients perform poorly with significant morbidity. Gene expression profiling has revealed that monopolar spindle 1 (MPS1) (TTK1) is highly expressed in medulloblastoma patient samples compared to that noted in normal cerebellum. MPS1 is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. The SAC can be activated in aneuploid cancer cells and MPS1 is overexpressed in many types of cancers. A previous study has demonstrated the effectiveness of inhibiting MPS1 with small-molecule inhibitors, but the role of MPS1 in medulloblastoma is unknown. In the present study, we demonstrated that MPS1 inhibition by shRNA or with a small-molecule drug, NMS-P715, resulted in decreased cell growth, inhibition of clonogenic potential and induction of apoptosis in cells belonging to both the Shh and group 3 medulloblastoma genomic signature. These findings highlight MPS1 as a rational therapeutic target for medulloblastoma.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive, fatal, childhood tumors that arise in the brainstem. DIPGs have no effective treatment, and their location and diffuse nature render them inoperable. Radiation therapy remains the only standard of care for this devastating disease. New therapeutic targets are needed to develop novel therapy for DIPG.
METHODS: We examined the expression of PLK1 mRNA in DIPG tumor samples through microarray analysis and found it to be up regulated versus normal pons. Using the DIPG tumor cells, we inhibited PLK1 using a clinically relevant specific inhibitor BI 6727 and evaluated the effects on, proliferation, apoptosis, induction of DNA damage and radio sensitization of the DIPG tumor cells.
RESULTS: Treatment of DIPG cell lines with BI 6727, a new generation, highly selective inhibitor of PLK1, resulted in decreased cell proliferation and a marked increase in cellular apoptosis. Cell cycle analysis showed a significant arrest in G2-M phase and a substantial increase in cell death. Treatment also resulted in an increased γH2AX expression, indicating induction of DNA damage. PLK1 inhibition resulted in radiosensitization of DIPG cells.
CONCLUSION: These findings suggest that targeting PLK1 with small-molecule inhibitors, in combination with radiation therapy, will hold a novel strategy in the treatment of DIPG that warrants further investigation.

Checkpoint kinase 1 (CHK1) is an integral component of the cell cycle as well as the DNA Damage Response (DDR) pathway. Previous work has demonstrated the effectiveness of inhibiting CHK1 with small-molecule inhibitors, but the role of CHK1 mediated DDR in medulloblastoma is unknown. CHK1, both at the mRNA and protein level, is highly expressed in medulloblastoma and elevated CHK1 expression in Group3 medulloblastoma is an adverse prognostic marker. CHK1 inhibition with the small-molecule drug AZD7762, results in decreased cell growth, increased DNA damage and cell apoptosis. Furthermore, AZD7762 acts in synergy with cisplatin in reducing cell proliferation in medulloblastoma. Similar phenotypic changes were observed with another CHK1 inhibitor, PF477736, as well as genetic knockdown using siRNA against CHK1. Treatments with small-molecule inhibitors of CHK1 profoundly modulated the expression of both upstream and downstream target proteins within the CHK1 signaling pathways. This suggests the presence of a feedback loop in activating CHK1. Overall, our results demonstrate that small-molecule inhibition of CHK1 in combination with, cisplatin, is more advantageous than either treatment alone, especially for Group 3 medulloblastoma, and therefore this combined therapeutic approach serves as an avenue for further investigation.

SOX10 is important in nonneoplastic oligodendroglial development, but mRNA transcripts and protein expression are identified in a wider variety of CNS glial neoplasms than oligodendrogliomas. We previously demonstrated high levels of SOX10 mRNA and protein in pilocytic astrocytomas (PAs) but not ependymomas (EPNs). We now extend these studies to investigate subsets of these 2 tumors that affect infants, pilomyxoid astrocytomas (PMAs) and infant (<1 year) ependymomas (iEPNs). By gene expression microarray analysis, we found that iEPNs and all EPNs in older children showed very low SOX10 expression levels, on average 7.1-fold below normal control tissues. EPN groups showed no significant difference in SOX10 expression between iEPN and EPN. PAs/PMAs had 24.1/29.4-fold higher transcript levels, respectively, than those in normal tissues. Using immunohistochemical analysis of adult, pediatric, and infantile EPNs and of PAs/PMAs, we found that EPNs from multiple anatomical locations and both age groups (n = 228) never showed 3+ diffuse nuclear immunostaining for SOX10; the majority were scored at 0 or 1+. Conversely, almost all pediatric and adult PAs and PMAs (n = 47) were scored as 3+. These results suggest that in select settings, SOX10 immunohistochemistry can supplement the diagnosis of PMA and PA and aid in distinguishing them from EPNs.

The current treatment for medulloblastoma includes surgical resection, radiation, and cytotoxic chemotherapy. Although this approach has improved survival rates, the high doses of chemotherapy required for clinical efficacy often result in lasting neurocognitive defects and other adverse events. Therefore, the development of chemosensitizing agents that allow dose reductions of cytotoxic agents, limiting their adverse effects but maintaining their clinical efficacy, would be an attractive approach to treat medulloblastoma. We previously identified WEE1 kinase as a new molecular target for medulloblastoma from an integrated genomic analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells and tissue. In addition, we demonstrated that WEE1 prevents DNA damage-induced cell death by cisplatin and that the WEE1 inhibitor AZD1775 displays synergistic activity with cisplatin. AZD1775 was developed as a WEE1 inhibitor from an initial hit from a high-throughput screen. However, given the lack of structure-activity data for AZD1775, we developed a small series of analogs to determine the requirements for WEE1 inhibition and further examine the effects of WEE1 inhibition in medulloblastoma. Interestingly, the compounds that inhibited WEE1 in the same nanomolar range as AZD1775 had significantly reduced single-agent cytotoxicity compared with AZD1775 and displayed synergistic activity with cisplatin in medulloblastoma cells. The potent cytotoxicity of AZD1775, unrelated to WEE1 inhibition, may result in dose-limiting toxicities and exacerbate adverse effects; therefore, WEE1 inhibitors that demonstrate low cytotoxicity could be dosed at higher concentrations to chemosensitize the tumor and potentiate the effect of DNA-damaging agents such as cisplatin.

INTRODUCTION: Pediatric adamantinomatous craniopharyngioma (ACP) is a histologically benign but clinically aggressive brain tumor that arises from the sellar/suprasellar region. Despite a high survival rate with current surgical and radiation therapy (75-95 % at 10 years), ACP is associated with debilitating visual, endocrine, neurocognitive and psychological morbidity, resulting in excheptionally poor quality of life for survivors. Identification of an effective pharmacological therapy could drastically decrease morbidity and improve long term outcomes for children with ACP.
RESULTS: Using mRNA microarray gene expression analysis of 15 ACP patient samples, we have found several pharmaceutical targets that are significantly and consistently overexpressed in our panel of ACP relative to other pediatric brain tumors, pituitary tumors, normal pituitary and normal brain tissue. Among the most highly expressed are several targets of the kinase inhibitor dasatinib - LCK, EPHA2 and SRC; EGFR pathway targets - AREG, EGFR and ERBB3; and other potentially actionable cancer targets - SHH, MMP9 and MMP12. We confirm by western blot that a subset of these targets is highly expressed in ACP primary tumor samples.
CONCLUSIONS: We report here the first published transcriptome for ACP and the identification of targets for rational therapy. Experimental drugs targeting each of these gene products are currently being tested clinically and pre-clinically for the treatment of other tumor types. This study provides a rationale for further pre-clinical and clinical studies of novel pharmacological treatments for ACP. Development of mouse and cell culture models for ACP will further enable the translation of these targets from the lab to the clinic, potentially ushering in a new era in the treatment of ACP.

Ependymoma (EPN) in childhood is a brain tumor with substantial mortality. Inflammatory response has been identified as a molecular signature of high-risk Group A EPN. To better understand the biology of this phenotype and aid therapeutic development, transcriptomic data from Group A and B EPN patient tumor samples, and additional malignant and normal brain data, were analyzed to identify the mechanism underlying EPN Group A inflammation. Enrichment of IL6 and STAT3 pathway genes were found to distinguish Group A EPN from Group B EPN and other brain tumors, implicating an IL6 activation of STAT3 mechanism. EPN tumor cell growth was shown to be dependent on STAT3 activity, as demonstrated using shRNA knockdown and pharmacologic inhibition of STAT3 that blocked proliferation and induced apoptosis. The inflammatory factors secreted by EPN tumor cells were shown to reprogram myeloid cells, and this paracrine effect was characterized by a significant increase in pSTAT3 and IL8 secretion. Myeloid polarization was shown to be dependent on tumor secretion of IL6, and these effects could be reversed using IL6-neutralizing antibody or IL6 receptor-targeted therapeutic antibody tocilizumab. Polarized myeloid cell production of IL8 drove unpolarized myeloid cells to upregulate CD163 and to produce a number of proinflammatory cytokines. Collectively, these findings indicate that constitutive IL6/STAT3 pathway activation is important in driving tumor growth and inflammatory cross-talk with myeloid cells within the Group A EPN microenvironment. Effective design of Group A-targeted therapy for children with EPN may require reversal of this potentially immunosuppressive and protumor pathway.

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

BACKGROUND: Smoking induces oncogenic TP53-mutations in head and neck squamous cell carcinomas (HNSCCs). Disruptive mutations of TP53-gene and expression of p16 protein [p16 (+)] in tumor tissue associate with worse and better prognosis, respectively. UDP-glucuronosyltransferase 2 family, polypeptide B17 (UGT2B17) detoxifies smoking-related metabolites. Differences among ethnic groups in UGT2B17 are extremely high. Homozygous deletions of UGT2B17 gene (UGT2B17-deletion) are a common copy number variant (CNV) among Japanese, but not a common CNV among Africans and Europeans. Thus, we examined Japanese patients with HNSCC to explore if UGT2B17-deletion and/or p16 (+) modify effects of smoking on TP53-mutations and affect relapse.
METHODS: We conducted a posthoc analysis of a prospective cohort. Polymerase chain reaction, immunohistochemistry, and direct sequencing were used to determine UGT2B17-deletion, p16 (+), and detailed TP53-mutations, respectively.
RESULTS: UGT2B17-deletion was observed in 80% of this study population. For this 80%, TP53-mutations were significantly more common among smokers than non-smokers (P = 0.0016), but this difference between smokers and nonsmokers was not significant for the 20% with UGT2B17. In patients with UGT2B17-deletion and p16 (+), simultaneously, TP53-mutations were much more common among smokers than among non-smokers (81% versus 17%; P = 0.0050). Patients with both UGT2B17-deletion and disruptive TP53-mutations had higher relapse rates than other patients (hazard ratio, 2.22; 95% confidence interval, 1.30 to 3.80, P = 0.004) in a stepwise method.
CONCLUSIONS: These results suggest that UGT2B17-deletion interacting with p16 (+) may modify effects of smoking on TP53-mutations and may further interact with the disruptive TP53-mutations to raise relapse rates among Japanese patients with HNSCC.

The heritability of colorectal cancer (CRC) is incompletely understood, and the contribution of undiscovered rare variants may be important. In search of rare disease-causing variants, we exome sequenced 22 CRC patients who were diagnosed before the age of 40 years. Exome sequencing data from 95 familial CRC patients were available as a validation set. Cases with known CRC syndromes were excluded. All patients were from Finland, a country known for its genetically homogenous population. We searched for rare nonsynonymous variants with allele frequencies below 0.1% in 3,374 Finnish and 58,112 non-Finnish controls. In addition, homozygous and compound heterozygous variants were studied. No genes with rare loss-of-function variants were present in more than one early-onset CRC patient. Three genes (ADAMTS4, CYTL1, and SYNE1) harbored rare loss-of-function variants in both early-onset and familial CRC cases. Five genes with homozygous variants in early-onset CRC cases were found (MCTP2, ARHGAP12, ATM, DONSON, and ROS1), including one gene (MCTP2) with a homozygous splice site variant. All discovered homozygous variants were exclusive to one early-onset CRC case. Independent replication is required to associate the discovered variants with CRC. These findings, together with a lack of family history in 19 of 22 (86%) early-onset patients, suggest genetic heterogeneity in unexplained early-onset CRC patients, thus emphasizing the requirement for large sample sizes and careful study designs to elucidate the role of rare variants in CRC susceptibility.

Inhaled xenobiotics such as tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone are mainly metabolized by phase I oxidase cytochrome P450, family 2, subfamily A, polypeptide 13 (CYP2A13), phase II conjugate UDP glucuronosyltransferase 2 family, polypeptide B17 (UGT2B17), and phase III transporter ATP-binding cassette, subfamily B (MDR/TAP), member 1 (ABCB1), with genetic polymorphisms implicated in lung cancer. Their genetic interaction and pulmonary expression regulation are largely unknown. We analyzed joint association for CYP2A13 and ABCB1 polymorphisms in 2 independent lung cancer case populations (669 and 566 patients) and 1 common control population (749 subjects), and characterized the trans-acting function of the lung development-related transcription factor forkhead box A2 (FOXA2). We undertook FOXA2 overexpression and down-regulation in lung epithelial cell lines, analyzed functional impact on the transactivation of CYP2A13, UGT2B17, and ABCB1, and measured correlation for their expressions in lung tissues. We found a substantial reduction in cancer risk (OR 0.39; 95% CI 0.25-0.61; Pinteraction = 0.029) associated with combined genotypes for CYP2A13 R257C and a functionary regulatory variant in the cis element of ABCB1 synergistically targeted by GATA binding protein 6 and FOXA2. Genetic manipulation of FOXA2 consistently influenced its binding to and transactivation of the promoters of CYP2A13, UGT2B17, and ABCB1, whose mRNA and protein expressions were all consistently correlated with those of FOXA2 in both tumorous and normal lung tissues. We therefore establish FOXA2 as a core transcriptional modulator for pulmonary xenobiotic metabolic pathways and uncover an etiologically relevant interaction between CYP2A13 and ABCB1, furthering our understanding of expression and function of the xenobiotic metabolism system.

Pilomyxoid astrocytomas (PMAs) manifest a more aggressive clinical course than pilocytic astrocytomas (PAs). Development of effective therapies demands a better biological understanding of PMA. We first conducted gene expression microarray analysis of 9 PMA and 13 PA from infra- and supratentorial sites. Unsupervised hierarchical clustering analysis demonstrated that tumors are grouped according to anatomic site, not diagnosis. Gene expression profiles were then contrasted between eight PMAs and six PAs, all supratentorial/hypothalamic/chiasmal. Clinical outcome of PMAs varied, with four out of four patients with diencephalic syndrome succumbing to disease, one of whom showed bulky metastatic leptomeningeal spread at autopsy, with bimodal maturation to PA in some areas and de-differentiation to glioblastoma in others. A surviving child has undergone multiple surgical debulking, with progressive maturation to PA over time. Ontology-enrichment analysis identified overexpression in PMAs of extracellular matrix and mitosis-related genes. Genes overexpressed in PMA vs. PA, ranked according to fold-change, included developmental genes H19, DACT2, extracellular matrix collagens (COL2A1; COL1A1) and IGF2BP3 (IMP3), the latter previously identified as an adverse prognostic factor in PMA and PA.

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

BACKGROUND: Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity.
METHODS: To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle-related kinases were associated with disease development. In addition a kinome-wide small interfering RNA (siRNA) screen was performed to identify kinases that, when inhibited, could prevent cell proliferation. The two genome-scale analyses were combined to identify key vulnerabilities in medulloblastoma. The inhibition of one of the identified targets was further investigated using RNAi and a small molecule inhibitor.
RESULTS: Combining the two analyses revealed that mitosis-related kinases were critical determinants of medulloblastoma cell proliferation. RNA interference (RNAi)-mediated knockdown of WEE1 kinase and other mitotic kinases was sufficient to reduce medulloblastoma cell proliferation. These data prompted us to examine the effects of inhibiting WEE1 by RNAi and by a small molecule inhibitor of WEE1, MK-1775, in medulloblastoma cell lines. MK-1775 inhibited the growth of medulloblastoma cell lines, induced apoptosis and increased DNA damage at nanomolar concentrations. Further, MK-1775 was synergistic with cisplatin in reducing medulloblastoma cell proliferation and resulted in an associated increase in cell death. In vivo MK-1775 suppressed medulloblastoma tumor growth as a single agent.
CONCLUSIONS: Taken together, these findings highlight mitotic kinases and, in particular, WEE1 as a rational therapeutic target for medulloblastoma.

Better understanding of ependymoma (EPN) biology at relapse is needed to improve therapy at this critical event. Convincing data exist defining transcriptionally distinct posterior fossa (PF) sub-groups A and B at diagnosis. The clinical and biological consequence of these sub-groups at recurrence has not yet been defined. Genome and transcriptome microarray profiles and clinical variables of matched primary and first recurrent PF EPN pairs were used to identify biologically distinct patterns of progression between EPN sub-groups at recurrence. Key findings were validated by histology and immune function assays. Transcriptomic profiles were partially conserved at recurrence. However, 4 of 14 paired samples changed sub-groups at recurrence, and significant sub-group-specific transcriptomic changes between primary and recurrent tumors were identified, which were predominantly immune-related. Further examination revealed that Group A primary tumors harbor an immune gene signature and cellular functionality consistent with an immunosuppressive phenotype associated with tissue remodeling and wound healing. Conversely, Group B tumors develop an adaptive, antigen-specific immune response signature and increased T-cell infiltration at recurrence. Clinical distinctions between sub-groups become more apparent after first recurrence. Group A tumors were more often sub-totally resected and had a significantly shorter time to subsequent progression and worse overall survival. Minimal tumor-specific genomic changes were observed for either PF Groups A or B at recurrence. Molecular sub-groups of PF EPN convey distinct immunobiologic signatures at diagnosis and recurrence, providing potential biologic rationale to their disparate clinical outcomes. Immunotherapeutic approaches may be warranted, particularly in Group A PF EPN.

Brainstem gangliogliomas (GGs), often cannot be resected, have a much poorer prognosis than those located in more common supratentorial sites and may benefit from novel therapeutic approaches. Therapeutically targetable BRAF c.1799T>A (p.V600E) (BRAF(V600E) ) mutations are harbored in roughly 50% of collective GGs taken from all anatomical sites. Large numbers of pediatric brainstem GGs, however, have not been specifically assessed and anatomic-and age-restricted assessment of genetic and biological factors are becoming increasingly important. Pediatric brainstem GGs (n = 13), non-brainstem GGs (n = 11) and brainstem pilocytic astrocytomas (PAs) (n = 8) were screened by standard Sanger DNA sequencing of BRAF exon 15. Five of 13 (38%) pediatric GG harbored a definitive BRAF(V600E) mutation, with two others exhibiting an equivocal result by this method. BRAF(V600E) was also seen in five of 11 (45%) non-brainstem GGs and one of eight (13%) brainstem PAs. VE1 immunostaining for BRAF(V600E) showed concordance with sequencing in nine of nine brainstem GGs including the two cases equivocal by Sanger. The equivocal brainstem GGs were subsequently shown to harbor BRAF(V600E) using a novel, more sensitive, RNA-sequencing approach, yielding a final BRAF(V600E) mutation frequency of 54% (seven of 13) in brainstem GGs. BRAF(V600E) -targeted therapeutics should be a consideration for the high percentage of pediatric brainstem GGs refractory to conventional therapies.

Despite increasing evidence that antitumor immune control exists in the pediatric brain, these findings have yet to be exploited successfully in the clinic. A barrier to development of immunotherapeutic strategies in pediatric brain tumors is that the immunophenotype of these tumors' microenvironment has not been defined. To address this, the current study used multicolor FACS of disaggregated tumor to systematically characterize the frequency and phenotype of infiltrating immune cells in the most common pediatric brain tumor types. The initial study cohort consisted of 7 pilocytic astrocytoma (PA), 19 ependymoma (EPN), 5 glioblastoma (GBM), 6 medulloblastoma (MED), and 5 nontumor brain (NT) control samples obtained from epilepsy surgery. Immune cell types analyzed included both myeloid and T cell lineages and respective markers of activated or suppressed functional phenotypes. Immune parameters that distinguished each of the tumor types were identified. PA and EPN demonstrated significantly higher infiltrating myeloid and lymphoid cells compared with GBM, MED, or NT. Additionally, PA and EPN conveyed a comparatively activated/classically activated myeloid cell-skewed functional phenotype denoted in particular by HLA-DR and CD64 expression. In contrast, GBM and MED contained progressively fewer infiltrating leukocytes and more muted functional phenotypes similar to that of NT. These findings were recapitulated using whole tumor expression of corresponding immune marker genes in a large gene expression microarray cohort of pediatric brain tumors. The results of this cross-tumor comparative analysis demonstrate that different pediatric brain tumor types exhibit distinct immunophenotypes, implying that specific immunotherapeutic approaches may be most effective for each tumor type.

Prognostic factors in pilocytic astrocytomas (PAs) and pilomyxoid astrocytomas (PMAs) include extent of resection, location, and age, but no molecular markers have been established. Insulin-like growth factor 2 mRNA binding protein 3 (IMP3, IGF2BP3) is predictive of an unfavorable prognosis in other tumors, including high-grade astrocytomas, but its role in PA/PMA is unknown. This study aimed to determine the expression and prognostic value of IMP3 in pediatric PA/PMAs. Insulin-like growth factor 2 mRNA binding protein 3 protein expression was examined by immunohistochemistry in 77 pediatric PAs (n = 70) and PMAs (n = 7) and scored on a subjective scale. Strong diffuse staining for IMP3 was observed in 31% (24 of 77) of tumors and associated with a shorter progression-free survival (hazard ratio, 2.63; p = 0.008). This cohort confirmed previously identified prognostic factors, including extent of resection, age, and tumor location. Currently, only clinical factors are weighed to stratify risk for patients and to identify those who should receive further therapy. Multivariate analyses identified IMP3 expression as an independent prognostic factor when combined with high-/low-risk stratification (hazard ratio, 2.45; p = 0.016). High IMP3, as assessed by immunohistochemistry, has potential use as an additional predictor of poor prognosis in pediatric PA/PMAs and warrants evaluation in larger cohorts.