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.

Rhabdoid tumors are caused by the deletion of SMARCB1, whose protein encodes the SMARCB1 subunit of the chromatin remodeling complex SWI/SNF that is involved in global chromatin organization and gene expression control. Simultaneously inhibiting the main players involved in the deregulated transcription machinery is a promising option for preventing exaggerated tumor cell proliferation and survival as it may bypass compensatory mechanisms. In support of this hypothesis, we report efficient impairment of cellular proliferation and strong induction of cell death elicited by inhibition of bromodomain protein BRD4 and transcription kinase CDK9 using small molecular compounds. Combination of both compounds efficiently represses antiapoptotic genes and the oncogene MYC. Our results provide a novel approach for the treatment of RT.

Ependymomas in children can arise throughout all compartments of the central nervous system (CNS). Highly malignant paediatric ependymoma subtypes are Group A tumours of the posterior fossa (PF-EPN-A) and RELA-fusion positive (ST-EPN-RELA) tumours in the supratentorial compartment. It was repeatedly reported in smaller series that accumulation of p53 is frequently observed in ependymomas and that immunohistochemical staining correlates with poor clinical outcome, while TP53 mutations are rare. Our TP53 mutation analysis of 130 primary ependymomas identified a mutation rate of only 3%. Immunohistochemical analysis of 398 ependymomas confirmed previous results correlating the accumulation of p53 with inferior outcome. Among the p53-positive ependymomas, the vast majority exhibited a RELA fusion leading to the hypothesis that p53 inactivation might be linked to RELA positivity.In order to assess the potential of p53 reactivation through MDM2 inhibition in ependymoma, we evaluated the effects of Actinomycin-D and Nutlin-3 treatment in two preclinical ependymoma models representing the high-risk subtypes PF-EPN-A and ST-EPN-RELA. The IC-50 of the agent as determined by metabolic activity assays was in the lower nano-molar range (0.2-0.7 nM). Transcriptome analyses of high-dose (100 nM), low-dose (5 nM) and non-treated cells revealed re-expression of p53 dependent genes including p53 upregulated modulator of apoptosis (PUMA) after low-dose treatment. At the protein level, we validated the Actinomycin-D induced upregulation of PUMA, and of p53 interaction partners MDM2 and p21. Proapoptotic effects of low-dose application of the agent were confirmed by flow cytometry. Thus, Actinomycin-D could constitute a promising therapeutic option for ST-EPN-RELA ependymoma patients, whose tumours frequently exhibit p53 inactivation.

Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy diagnosed in women under age 40. We and others recently determined that germline and/or somatic deleterious mutations in SMARCA4 characterize SCCOHT. Alterations in this gene, or the related SWI/SNF chromatin remodeling gene SMARCB1, have been previously reported in atypical teratoid/rhabdoid tumors (ATRTs) and malignant rhabdoid tumors (MRTs). To further describe the somatic landscape of SCCOHT, we performed whole exome sequencing on 14 tumors and their matched normal tissues and compared their genomic alterations with those in ATRT and ovarian high grade serous carcinoma (HGSC). We confirmed that SMARCA4 is the only recurrently mutated gene in SCCOHT, and show that recurrent allelic imbalance is observed exclusively on chromosome 19p, where SMARCA4 resides. By comparing genomic alterations between SCCOHT, ATRT and HGSC, we demonstrate that SCCOHTs, like ATRTs, have a remarkably simple genome and harbor significantly fewer somatic protein-coding mutations and chromosomal alterations than HGSC. Furthermore, a comparison of global DNA methylation profiles of 45 SCCOHTs, 65 ATRTs, and 92 HGSCs demonstrates a strong epigenetic correlation between SCCOHT and ATRT. Our results further confirm that the genomic and epigenomic signatures of SCCOHT are more similar to those of ATRT than HGSC, supporting our previous hypothesis that SCCOHT is a rhabdoid tumor and should be renamed MRT of the ovary. Furthermore, we conclude that SMARCA4 inactivation is the main cause of SCCOHT, and that new distinct therapeutic approaches should be developed to specifically target this devastating tumor.

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1-/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1-/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

Atypical teratoid rhabdoid tumor (ATRT) is an aggressive and malignant pediatric brain tumor. Polo-like kinase 1 (PLK1) is highly expressed in many cancers and essential for mitosis. Overexpression of PLK1 promotes chromosome instability and aneuploidy by overriding the G2-M DNA damage and spindle checkpoints. Recent studies suggest that targeting PLK1 by small molecule inhibitors is a promising approach to tumor therapy. We investigated the effect of PLK1 inhibition in ATRT. Gene expression analysis showed that PLK1 was overexpressed in ATRT patient samples and tumor cell lines. Genetic inhibition of PLK1 with shRNA potently suppressed ATRT cell growth in vitro. Treatment with the PLK1 inhibitor BI 6727 (Volasertib) significantly decreased cell growth, inhibited clonogenic potential, and induced apoptosis. BI6727 treatment led to G2-M phase arrest, consistent with PLK1's role as a critical regulator of mitosis. Moreover, inhibition of PLK1 by BI6727 suppressed the tumor-sphere formation of ATRT cells. Treatment also significantly decreased levels of the DNA damage proteins Ku80 and RAD51 and increased γ-H2AX expression, indicating that BI 6727 can induce DNA damage. Importantly, BI6727 significantly enhanced radiation sensitivity of ATRT cells. In vivo, BI6727 slowed growth of ATRT tumors and prolonged survival in a xenograft model. PLK1 inhibition is a compelling new therapeutic approach for treating ATRT, and the use of BI6727 should be evaluated in clinical studies.