Highlights of children with Cancer UK’s workshop on drug delivery in paediatric brain tumours

The first Workshop on Drug Delivery in Paediatric Brain Tumours was hosted in London by the charity Children with Cancer UK. The goals of the workshop were to break down the barriers to treating central nervous system (CNS) tumours in children, leading to new collaborations and further innovations in this under-represented and emotive field. These barriers include the physical delivery challenges presented by the blood–brain barrier, the underpinning reasons for the intractability of CNS cancers, and the practical difficulties of delivering cancer treatment to the brains of children. Novel techniques for overcoming these problems were discussed, new models brought forth, and experiences compared

Intratumoral Genetic and Functional Heterogeneity in Pediatric Glioblastoma.

Pediatric glioblastoma (pGBM) is a lethal cancer with no effective therapies. To understand the mechanisms of tumor evolution in this cancer, we performed whole-genome sequencing with linked reads on longitudinally resected pGBM samples. Our analyses showed that all diagnostic and recurrent samples were collections of genetically diverse subclones. Clonal composition rapidly evolved at recurrence, with less than 8% of nonsynonymous single-nucleotide variants being shared in diagnostic-recurrent pairs. To track the origins of the mutational events observed in pGBM, we generated whole-genome datasets for two patients and their parents. These trios showed that genetic variants could be (i) somatic, (ii) inherited from a healthy parent, or (iii) de novo in the germlines of pGBM patients. Analysis of variant allele frequencies supported a model of tumor growth involving slow-cycling cancer stem cells that give rise to fast-proliferating progenitor-like cells and to nondividing cells. Interestingly, radiation and antimitotic chemotherapeutics did not increase overall tumor burden upon recurrence. These findings support an important role for slow-cycling stem cell populations in contributing to recurrences, because slow-cycling cell populations are expected to be less prone to genotoxic stress induced by these treatments and therefore would accumulate few mutations. Our results highlight the need for new targeted treatments that account for the complex functional hierarchies and genomic heterogeneity of pGBM. SIGNIFICANCE: This work challenges several assumptions regarding the genetic organization of pediatric GBM and highlights mutagenic programs that start during early prenatal development.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2111/F1.large.jpg.Wellcome Trust Royal Societ

TERT promoter mutations are highly recurrent in SHH subgroup medulloblastoma

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 wit

Cytogenetic Prognostication Within Medulloblastoma Subgroups

PURPOSE: Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication. PATIENTS AND METHODS: Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models. RESULTS: Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas. CONCLUSION: Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials

Regulation Of Anti-Oxidant and Anti-Apoptotic Genes By Progesterone in Cardiomyocytes

The anthracycline quinone, doxorubicin (Adriamycin) is an antineoplastic agent that has substantial therapeutic activity against a broad variety of human cancers. Unfortunately, the use of this agent is limited by its cardiac toxicity, which is associated with free radical formation leading to apoptotic cell death. The goal of this work is to improve our understanding about doxorubicin induced cardiomyopathy and to identify compounds to limit doxorubicin induced cardiomyopathy. The knowledge gained here will have a generalized impact on all cardiac diseases involving oxidative stress and apoptosis. We show that doxorubicin induced apoptosis in primary neonatal rat cardiomyocytes can be attenuated by progesterone (PG). The anti-apoptotic action of PG was blocked by a progesterone receptor antagonist, Mifepristone (MF), indicating a progesterone receptor dependent pathyway. Affymetrix gene analyses found that PG treated cardiomyocytes increased the expression of 180 genes. Among the genes upregulated is NAD(P)H: Quinone Oxidoreductase-1 (NQO1) gene. NQO1 is a flavo-enzyme that can catalyze a two-electron reduction of Dox to a more stable hydroquinone, thereby acting as a defense mechanism against oxidative stress. The induction of NQO1 mRNA and NQO1 activity in cardiomyocytes was observed in a dose and time-dependent manner with PG treatment and was blocked by MF. Induction of NQO1 by b-naphoflavone, an inducer of NQO1, resulted in a decrease in caspase-3 activity. However, inhibition of NQO1 by dicoumarol did not attenuate the cytoprotective effect of PG. This data indicates that although induction of NQO1 can decrease Dox induced apoptosis, this is not the primary mechanism of cytoprotection induced by PG. Microarray analyses revealed that PG induced an increase of Bcl-XL mRNA. Inhibiting the expression of Bcl-XL using siRNA reduced the anti-apoptotic effect of PG, suggesting that Bcl-XL is a key player in PG induced cytoprotection. Western blot analyses indicated that PG induced the expression of Bcl-XL in a dose and time dependent manner consistent with the protective effect of PG. Induction of Bcl-XL by PG was blocked by cyclohexamide, but was not blocked by Actinomycin D indicating that a transcriptionally independent mechanism is responsible for the induction of Bcl-XL by PG. The activity of a bcl-x 3'UTR reporter was induced by PG and blocked by MF. These data suggest that PG may induce stabilization of the Bcl-X mRNA. We further explored the mechanism of PG induced Bcl-XL gene expression by comparing the effect of PG to two other steroids: corticosterone (CT) and retinoic acid (RA). Both CT and RA attenuate Dox induced apoptosis in cardiomyocytes. CT, but not RA or PG induced the activity of a GRE reporter plasmid. Analysis of the 5' region of the Bcl-XL promoter indicated that RA and CT, but not PG induced the activity of the 0.9kb region of the Bcl-XL promoter. The induction of the 0.9kb reporter plasmid by CT was glucocorticoid receptor dependent, since it was inhibited by MF. The Bcl-XL promoter does not contain any glucocorticoid or retinoid response elements, but does have AP-1 and NFkB response elements. CT, but not RA or PG induced the activity of an AP-1 reporter plasmid. RA, but not CT or PG induced the activity of an NFkB reporter plasmid. The induction of the 0.9kb Bcl-XL reporter plasmid by CT was blocked by expression of a dominant negative c-jun, TAM67 as well SB202190 indicating a nongenomic effect of CT in activating the Bcl-XL promoter through a p38 MAPK mediated AP-1 mechanism. Therefore although all three types of nuclear receptor ligands induce bcl-xL expression, the effect of CT is mediated by transcriptional activation by AP-1 signaling while NF-kB transcription factor appears to be involved in RA indced bcl-xL transcription

Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress.

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