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 choosing therapies based on assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic make-up of this brain cancer with nearly 80% harboring a K27M-H3.3 or K27M-H3.1 mutation. 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 are three molecularly distinct subgroups (H3-K27M, Silent, MYCN) and uncovering a novel recurrent activating mutation in the activin receptor ACVR1, in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement.

National Cancer Institute-National Institutes of Health, Canadian Institutes of Health Research, German Research Foundation, Canadian Retinoblastoma Society, Hyland Foundation, Toronto Netralaya and Doctors Lions Clubs, Ontario Ministry of Health and Long Term Care, UK-Essen, and Foundations Avanti-STR and KiKa.

Because of the large number of growth-regulated genes containing binding sites for the transcription factors Sp1 and E2F and the reported ability of E2F to mediate cell cycle (growth) regulation, we studied interactions between E2F1 and Sp1. In transient transfection assays using Drosophila melanogaster SL2 cells, transfection with both Sp1 and E2F1 expression vectors resulted in greater than 85-fold activation of transcription from a hamster dihydrofolate reductase reporter construct, whereas cotransfection with either the Sp1 or E2F1 expression vector resulted in 30-or <2-fold activation, respectively. Therefore, these transcription factors act synergistically in activation of dihydrofolate reductase transcription. Transient transfection studies demonstrated that E2F1 could superactivate Sp1-dependent transcription in a promoter containing only Sp1 sites and that Sp1 could superactivate transcription of promoters through E2F sites, further demonstrating that these factors functionally interact with one another. Coimmunoprecipitation studies revealed that Sp1 and E2F1 are physically associated in Drosophila cells transfected with Sp1 and E2F1 expression vectors and in human cells, with maximal interaction detected in mid-to late G 1 . Additionally, E2F1 and Sp1 interact in vitro through specific domains of each protein, and the physical interaction and functional synergism appear to require the same regions. Taken together, these data demonstrate that E2F1 and Sp1 both functionally and physically interact; therefore, through this interaction, Sp1 and E2F1 may regulate transcription of genes containing binding sites for either or both factors.

The novel oncogene KIF14 (kinesin family member 14) shows genomic gain and overexpression in many cancers including OvCa (ovarian cancer). We discovered that expression of the mitotic kinesin KIF14 is predictive of poor outcome in breast and lung cancers. We now determine the prognostic significance of KIF14 expression in primary OvCa tumors, and evaluate KIF14 action on OvCa cell tumorigenicity in vitro. Gene-specific multiplex PCR and real-time QPCR were used to measure KIF14 genomic (109 samples) and mRNA levels (122 samples) in OvCa tumors. Association of KIF14 with clinical variables was studied using Kaplan-Meier survival and Cox regression analyses. Cellular effects of KIF14 overexpression (stable transfection) and inhibition (stable shRNA knockdown) were studied by proliferation (cell counts), survival (Annexin V immunocytochemistry) and colony formation (soft-agar growth). KIF14 genomic gain (>2.6 copies) was present in 30% of serous OvCas, and KIF14 mRNA was elevated in 91% of tumors versus normal epithelium. High KIF14 in tumors independently predicted for worse outcome (p 5 0.03) with loss of correlation with proliferation marker expression and increased rates of recurrence. Overexpression of KIF14 in OvCa cell lines increased proliferation and colony formation (p < 0.01), whereas KIF14 knockdown induced apoptosis and dramatically reduced colony formation (p < 0.05). Our findings indicate that KIF14 mRNA is an independent prognostic marker in serous OvCa. Dependence of OvCa cells on KIF14 for maintenance of in vitro colony formation suggests a role of KIF14 in promoting a tumorigenic phenotype, beyond its known role in proliferation.Ovarian Cancer (OvCa) is the leading cause of death from gynecological malignancies in the Western World. 1 The majority of women who present with epithelial OvCa will die of their disease because of diagnosis at advanced stage. Treatment of OvCa has been virtually unchanged in over 40 years and includes cytoreductive surgery and cytotoxic chemotherapy. Improved surgical techniques, new chemotherapeutic agents, altered modes of chemotherapy delivery and emerging molecular targets have improved life expectancy but have done little to impact cure rates. There is a desperate need for improved early detection, diagnostic and prognostic indicators, and the identification of molecular pathways that drive and maintain OvCa cells, to achieve novel therapies that can cure.Our studies of the pediatric eye cancer retinoblastoma revealed some fundamental mechanisms of cancer initiation and development. [2][3][4][5][6][7][8] The most prevalent karyotypic abnormality in retinoblastoma following RB1 loss is gain of the long arm of chromosome 1. 4,5 Subsequently, 1q gain was shown to be common in breast, lung, liver, papillary renal cell and OvCas. [9][10][11][12][13][14][15][16][17][18][19] Expression analysis of genes within the minimal 1q region of gain (1q32.1) revealed the mitotic kinesin family member 14 (KIF14) as the potential oncogene. 13 We showed that high KIF14 expression in ...

Paediatric brain tumours arising in the thalamus present significant diagnostic and therapeutic challenges to physicians due to their sensitive midline location. As such, genetic analysis for biomarkers to aid in the diagnosis, prognosis and treatment of these tumours is needed. Here, we identified 64 thalamic gliomas with clinical follow-up and characterized targeted genomic alterations using newly optimized droplet digital and NanoString-based assays. The median age at diagnosis was 9.25 years (range, 0.63–17.55) and median survival was 6.43 (range, 0.01–27.63) years. Our cohort contained 42 and 22 tumours reviewed as low and high grade gliomas, respectively. Five (12 %) low grade and 11 (50 %) high grade gliomas were positive for the H3F3A/HIST1H3B K27M (H3K27M) mutation. Kaplan-Meier survival analysis revealed significantly worse overall survival for patients harbouring the H3K27M mutation versus H3F3A/HIST1H3B wild type (H3WT) samples (log-rank p < 0.0001) with a median survival of 1.02 vs. 9.12 years. Mitogen-activated protein kinase (MAPK) pathway activation via BRAF or FGFR1 hotspot mutations or fusion events were detected in 44 % of patients, and was associated with long-term survival in the absence of H3K27M (log-rank p < 0.0001). Multivariate analysis demonstrated H3K27M status and high grade histology to be the most significant independent predictors of poor overall survival with hazard ratios of 6.945 and 7.721 (p < 0.0001), respectively. In contrast, MAPK pathway activation is a predictor of favourable patient outcome, although not independent of other clinical factors. Importantly, we show that low grade malignancies may harbour H3K27M mutations and that these tumours show a dismal survival compared to low grade H3WT cases. Our data strongly supports the inclusion of targeted genetic testing in childhood thalamic tumours to most accurately stratify patients into appropriate risk groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0353-0) contains supplementary material, which is available to authorized users.

The transcription factor E2F and its regulation by pRB and related pocket proteins are central to cell cycle control in higher eukaryotes. Much of our knowledge of this regulation has come from studies using immediate-early proteins of DNA tumor viruses. Previously, we reported that the 72-kDa immediate-early region 1 gene product of the human cytomegalovirus, IE72, transactivates the dihydrofolate reductase promoter through the E2F site and that it physically interacts with E2F1 (M. J. Margolis, S. Pajovic, E. L. Wong, M. Wade, R. Jupp, J. A. Nelson, and J. C. Azizkhan, J. Virol. 69:7759-7767, 1995). In this study, we further characterized the mechanism by which IE72 modulates E2F-dependent transcription. In vitro phosphorylation reactions using gel-purified bacterially expressed proteins revealed that IE72 is a kinase that autophosphorylates and phosphorylates E2F1, -2, and -3 (but not E2F4 or -5) and the RB-related pocket proteins p130 and p107 (but not pRB). The region of IE72 spanning amino acids 173 to 197 shows a high level of homology to the ATP binding sites in over 500 kinases. The kinase-negative protein IE72⌬ATP, from which this region has been deleted, cannot activate E2F-dependent transcription. The kinase activity of IE72 is also required for its ability to reduce the association of E2F4 with p107 and p130. Taken together, these data suggest that the kinase activity of IE72 is required for E2F-dependent transcriptional activation and that this is likely to result from phosphorylation of specific members of the E2F and pocket protein families by IE72.E2F was originally identified as a transcription factor required by E1A for activation of the adenovirus E2 promoter. The gene encoding dihydrofolate reductase (DHFR) was the first cellular gene shown to contain a binding site for E2F (6), and transactivation of its promoter by adenovirus E1A was found to be E2F dependent (18). Subsequently, E2F was shown to be involved in the cell cycle regulation of several genes important in cellular growth control (for reviews, see references 23, 24, and 38). E2F activity is regulated by its interaction with the product of the tumor suppressor retinoblastoma susceptibility gene, pRB, and the related pocket proteins, p107 and p130, which are themselves tightly regulated during differentiation (36) and the cell cycle (for a review, see reference 32). With the cloning of E2F1, followed by the cloning of other, related proteins, E2F was shown to consist of a family with five members that heterodimerize with DP1 or DP2, which are members of a related protein family (for a review, see reference 23). The different E2F family members show specificity in their interactions with the pocket proteins; whereas E2F4 is able to interact with all of the pocket proteins, E2F1, -2, and -3 appear to interact only with pRB and E2F5 apparently interacts only with p130 (4,13,16,19,21,25,29). Modulation of transcription from promoters containing E2F sites can be achieved through increased E2F transactivation activity by release of free E...

Alkylating agents are a frontline therapy for the treatment of several aggressive cancers including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed; increasing therapeutic response while minimizing toxicity. Using a siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM) were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors.