Jessica I. Hoell

RNA targets of wild-type and mutant FET family proteins

FUS, EWSR1 and TAF15, constituting the FET protein family, are abundant, highly conserved RNA-binding proteins with important roles in oncogenesis and neuronal disease, yet their RNA targets and recognition elements are unknown. Using PAR-CLIP, we defined global RNA targets for all human FET proteins and two ALS-causing human FUS mutants. FET members showed similar binding profiles, whereas FUS mutants showed a drastically altered binding pattern, consistent with changes in subcellular localization.

Comprehensive Analysis of Long Non-Coding RNAs in Ovarian Cancer Reveals Global Patterns and Targeted DNA Amplification

Long non-coding RNAs (lncRNAs) are emerging as potent regulators of cell physiology, and recent studies highlight their role in tumor development. However, while established protein-coding oncogenes and tumor suppressors often display striking patterns of focal DNA copy-number alteration in tumors, similar evidence is largely lacking for lncRNAs. Here, we report on a genomic analysis of GENCODE lncRNAs in high-grade serous ovarian adenocarcinoma, based on The Cancer Genome Atlas (TCGA) molecular profiles. Using genomic copy-number data and deep coverage transcriptome sequencing, we derived dual copy-number and expression data for 10,419 lncRNAs across 407 primary tumors. We describe global correlations between lncRNA copy-number and expression, and associate established expression subtypes with distinct lncRNA signatures. By examining regions of focal copy-number change that lack protein-coding targets, we identified an intergenic lncRNA on chromosome 1, OVAL, that shows narrow focal genomic amplification in a subset of tumors. While weakly expressed in most tumors, focal amplification coincided with strong OVAL transcriptional activation. Screening of 16 other cancer types revealed similar patterns in serous endometrial carcinomas. This shows that intergenic lncRNAs can be specifically targeted by somatic copy-number amplification, suggestive of functional involvement in tumor initiation or progression. Our analysis provides testable hypotheses and paves the way for further study of lncRNAs based on TCGA and other large-scale cancer genomics datasets.

Recurrent RHOA Mutations in Pediatric Burkitt Lymphoma Treated According to the NHL-BFM Protocols

Burkitt lymphoma (BL) is the most frequent B‐cell lymphoma in childhood. Genetically, it is characterized by the presence of an IG‐MYC translocation which is supposed to be an initiating but not sufficient event in Burkitt lymphomagenesis. In a recent whole‐genome sequencing study of four cases, we showed that the gene encoding the ras homolog family member A (RHOA) is recurrently mutated in pediatric BL. Here, we analyzed RHOA by Sanger sequencing in a cohort of 101 pediatric B‐cell lymphoma patients treated according to Non‐Hodgkins Lymphoma Berlin–Frankfurt–Münster (NHL‐BFM) study protocols. Among the 78 BLs in this series, an additional five had RHOA mutations resulting in a total incidence of 7/82 (8.5%) with c.14G>A (p.R5Q) being present in three cases. Modeling the mutational effect suggests that most of them inactivate the RHOA protein. Thus, deregulation of RHOA by mutation is a recurrent event in Burkitt lymphomagenesis in children.

MINCR is a MYC-induced lncRNA able to modulate MYC’s transcriptional network in Burkitt lymphoma cells

Significance Gains of the MYC gene are the most common imbalances in cancer and are associated with poor prognosis, particularly in B-cell lymphoma. Recent advances in DNA sequencing have revealed the existence of thousands of long noncoding RNAs (lncRNAs) with unknown functional relevance. We have here identified a MYC-regulated lncRNA that we named MYC-induced long noncoding RNA (MINCR) that has a strong correlation with MYC expression in cancer. We show that MINCR is functional and controls cell cycle progression by influencing the expression of MYC-regulated cell cycle genes. MINCR is, therefore, a novel player in MYC’s transcriptional network, with the potential to open new therapeutic windows in the fight against malignant lymphoma and, possibly, all cancers that rely on MYC expression. Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”, we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.

Symptoms and management of pediatric patients with incurable brain tumors in palliative home care

INTRODUCTION Brain tumors have the highest disease-related mortality rate of all pediatric cancers. The goal of this study was to determine whether all children with incurable brain tumors cared for by a pediatric palliative care team in a home setting suffer from the same symptoms towards the end of their lives or whether there are differences between the tumor localizations with implications for palliative care. PATIENTS AND METHODS This study was conducted as a retrospective, single center chart review including all patients treated between January 1st 2000 and December 31st 2013. RESULTS 70 children, adolescents and young adults were included in the analysis. Symptom burden was high with a mean number of symptoms of 7.2 per patient. 74% of the symptoms already existed one week before death. Within the last week of life, impaired consciousness (75.7%) most often occurred. Furthermore, symptoms considerably depended on tumor localization. Patients with supratentorial tumors presented more frequently with seizures (p < 0.05), coma (p < 0.01), nausea and emesis (p < 0.01). Ataxia (p < 0.001) occurred most frequently in infratentorial tumors and speech disturbances (p < 0.05), cranial nerve paralysis (p < 0.001), and tetraparesis (p < 0.001) in brain stem tumors. 84.3% of the patients needed analgesics, only 64.4% WHO class III analgesics. Anticonvulsants were given more often in supratentorial tumors (p < 0.01). CONCLUSIONS Caring for a dying child suffering from a brain tumor needs increased awareness of the neurological deterioration. The symptom pattern strongly depends on the tumor localization and significantly differs between supratentorial, infratentorial and brain stem tumors.

The cell cycle regulator CCDC6 is a key target of RNA-binding protein EWS.

Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma.

Biochemical and bioinformatic methods for elucidating the role of RNA–protein interactions in posttranscriptional regulation

Abstract Our understanding of transcriptional gene regulation has dramatically increased over the past decades, and many regulators of gene expression, such as transcription factors, have been analyzed extensively. Additionally, in recent years, deeper insights into the physiological roles of RNA have been obtained. More precisely, splicing, polyadenylation, various modifications, localization and the translation of messenger RNAs (mRNAs) are regulated by their interaction with RNA-binding proteins (RBPs). New technologies now enable the analysis of this regulation at different levels. A technique known as ultraviolet (UV) cross-linking and immunoprecipitation (CLIP) allows us to determine physical protein–RNA interactions on a genome-wide scale. UV cross-linking introduces covalent bonds between interacting RBPs and RNAs. In combination with immunoprecipitation and deep sequencing techniques, tens of millions of short reads (representing bound RNAs by an RBP of interest) are generated and are used to characterize the regulatory network mediated by an RBP. Other methods, such as mass spectrometry, can also be used for characterization of cross-linked RBPs and RNAs instead of CLIP methods. In this review, we discuss experimental and computational methods for the generation and analysis of CLIP data. The computational methods include short-read alignment, annotation and RNA-binding motif discovery. We describe the challenges of analyzing CLIP data and indicate areas where improvements are needed.

Genomic profiling of Acute lymphoblastic leukemia in ataxia telangiectasia patients reveals tight link between ATM mutations and chromothripsis

Recent developments in sequencing technologies led to the discovery of a novel form of genomic instability, termed chromothripsis. This catastrophic genomic event, involved in tumorigenesis, is characterized by tens to hundreds of simultaneously acquired locally clustered rearrangements on one chromosome. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, an essential guardian of genome stability, would show a higher prevalence of chromothripsis due to the associated defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) arising in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared with tumors from individuals with other types of DNA repair syndromes (27 cases total, 10 with Ataxia Telangiectasia). Our data suggest that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients may be linked with frequent chromothripsis. Furthermore, we show that ATM loss is associated with increased chromothripsis prevalence in additional tumor entities.

Outcome of relapse after allogeneic HSCT in children with ALL enrolled in the ALL‐SCT 2003/2007 trial

Relapse remains the major cause of treatment failure in children with high‐risk acute lymphoblastic leukaemia (ALL) undergoing allogeneic haematopoietic stem‐cell transplantation (allo‐SCT). Prognosis is considered dismal but data on risk factors and outcome are lacking from prospective studies. We analysed 242 children with recurrence of ALL after first allo‐SCT enrolled in the Berlin‐Frankfurt‐Munster (BFM) ALL‐SCT‐BFM 2003 and ALL‐SCT‐BFM international 2007 studies. Median time from allo‐SCT to relapse was 7·7 months; median follow‐up from relapse after allo‐SCT until last follow‐up was 3·4 years. The 3‐year event‐free survival (EFS) was 15% and overall survival (OS) was 20%. The main cause of death was disease progression or relapse (86·5%). The majority of children (48%) received salvage therapy without second allo‐SCT, 26% of the children underwent a second allo‐SCT and 25% received palliative treatment only. In multivariate analyses, age, site of relapse, time to relapse and type of salvage therapy were identified as significant prognostic factors for OS and EFS, whereas factors associated with first SCT were not statistically significant. Combined approaches incorporating novel immunotherapeutic treatment options and second allo‐SCT hold promise to improve outcome in children with post allo‐SCT relapse.