Suzanne Vanhauwaert

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

The Notch driven long non-coding RNA repertoire in T-cell acute lymphoblastic leukemia

Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling. For this purpose, we measured the transcriptional response of all protein coding genes and long non-coding RNAs upon pharmacological Notch inhibition in the human T-cell acute lymphoblastic leukemia cell line CUTLL1 using RNA-sequencing. Similar Notch dependent profiles were established for normal human CD34+ thymic T-cell progenitors exposed to Notch signaling activity in vivo. In addition, we generated long non-coding RNA expression profiles (array data) from ex vivo isolated Notch active CD34+ and Notch inactive CD4+CD8+ thymocytes and from a primary cohort of 15 T-cell acute lymphoblastic leukemia patients with known NOTCH1 mutation status. Integration of these expression datasets with publicly available Notch1 ChIP-sequencing data resulted in the identification of long non-coding RNAs directly regulated by Notch activity in normal and malignant T cells. Given the central role of Notch in T-cell acute lymphoblastic leukemia oncogenesis, these data pave the way for the development of novel therapeutic strategies that target hyperactive Notch signaling in human T-cell acute lymphoblastic leukemia.

Refinement of the critical 2p25.3 deletion region: the role of MYT1L in intellectual disability and obesity

Purpose:Submicroscopic deletions of chromosome band 2p25.3 are associated with intellectual disability and/or central obesity. Although MYT1L is believed to be a critical gene responsible for intellectual disability, so far no unequivocal data have confirmed this hypothesis.Methods:In this study we evaluated a cohort of 22 patients (15 sporadic patients and two families) with a 2p25.3 aberration to further refine the clinical phenotype and to delineate the role of MYT1L in intellectual disability and obesity. In addition, myt1l spatiotemporal expression in zebrafish embryos was analyzed by quantitative polymerase chain reaction and whole-mount in situ hybridization.Results:Complete MYT1L deletion, intragenic deletion, or duplication was observed in all sporadic patients, in addition to two patients with a de novo point mutation in MYT1L. The familial cases comprise a 6-Mb deletion in a father and his three children and a 5′ MYT1L overlapping duplication in a father and his two children. Expression analysis in zebrafish embryos shows specific myt1l expression in the developing brain.Conclusion:Our data strongly strengthen the hypothesis that MYT1L is the causal gene for the observed syndromal intellectual disability. Moreover, because 17 patients present with obesity/overweight, haploinsufficiency of MYT1L might predispose to weight problems with childhood onset.Genet Med 17 6, 460–466.

Expressed Repeat Elements Improve RT-qPCR Normalization across a Wide Range of Zebrafish Gene Expression Studies

The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.

Expressed repetitive elements are broadly applicable reference targets for normalization of reverse transcription-qPCR data in mice

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data.

Abstract 4886: The BRIP1 DNA helicase is a 17q dosage sensitive cooperative driver in neuroblastoma

Neuroblastoma (NB) is an aggressive pediatric tumor arising from sympathetic neuronal progenitors. NBs have a low mutation burden while copy number alterations are highly recurrent: MYCN amplification is present in half of high risk tumors often accompanied by 1p deletions while MYCN non-amplified aggressive NB frequently exhibit 11q deletions. Remarkably, both high risk groups show almost invariably chromosome 17q gain and we also reported that the mouse syntenic chromosome 11 region was gained during MYCN driven tumor formation. We propose that one or more dosage sensitive genes on 17q act as cooperative drivers during NB development. Using an integrated bioinformatics analysis, we identified several candidate drivers implicated in DNA repair including BRIP1, also known as FANCJ and located on 17q23.2. BRIP1 acts as a DNA helicase in unwinding of stable G-quadruplex (G4) structures in single stranded DNA during replication ensuring timely progression through S-phase. We show that BRIP1 knock down causes increased replicative stress in MYCN overexpressing NB cells as evidenced as shown by increased RPA32 levels and reduced replication fork velocity. Overexpression of BRIP1 in dβh-MYCN-eGFP transgenic zebrafish caused accelerated tumor formation supporting its role as cooperative driver gene. Gene expression profiling after BRIP1 knock down confirmed enrichment for gene sets implicated in DNA replication and repair and are indicative for perturbation of G4 enriched genes. We also identified further additional 17q dosage sensitive genes implicated in replication fork dynamics including BRCA1, BRCA2, EME1 and TOP2A. We propose that 17q gain acts as an amplifier for expression of multiple genes implicated in control of replicative stress and replication fork dynamics. Finally, we explored whether this replicative stress resistance phenotype could represent a novel therapeutic vulnerability for NB cells or other MYC(N) driven tumor entities. To this end, we tested several compounds for synergistic interaction with G4 stabilizing ligands such as TMPYP4, pyridostatin and BRACO-19 with promising results. In conclusion, we identified BRIP1 as 17q cooperative driver in NB through relief from G4 induced replication fork stalling in rapidly dividing tumor cells. Further, NB cells exhibit replicative stress resistance through upregulation of multiple critical regulators of replication fork dynamics, offering a new venue for therapeutic interventions. Citation Format: Suzanne Vanhauwaert, Kaat Durinck, Els Janssens, Givani Dewyn, Bram De Wilde, Genevieve Laureys, Daniel Carter, Chueng Belamy, Katleen De Preter, Christophe Vanneste, Frank Speleman. The BRIP1 DNA helicase is a 17q dosage sensitive cooperative driver in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4886. doi:10.1158/1538-7445.AM2017-4886

Abstract 1527: BRD3 as a specific vulnerable therapeutic target in neuroblastoma

Introduction: BET inhibitors have raised high expectations for cancer treatment given their anti-proliferative effect by inhibiting BRD4 controlled enhancer activity of highly transcribed genes such as MYC(N). However, current inhibitors also target BRD2 and BRD3 which are functionally nonredundant with BRD4. In neuroblastoma only MYCN amplified tumors respond well to these drugs. Methods: We performed an integrated bioinformatics approach to scrutinize BET family genes as well as further candidate epigenetic regulators as targets for novel therapies in neuroblastoma. Results: First we performed a time-resolved expression data analysis of week 1 and 2 hyperplastic lesions and tumors derived from the TH-MYCN transgenic mouse model and confirmed dynamic regulation during tumor development for established neuroblastoma oncogenes and tumor suppressor genes. Next, we filtered within the highest upregulated genes for Cancer Gene Census (CGC) genes and identified 21 upregulated CGC genes mainly involved in chromatin remodeling and DNA repair. Finally, after further selection based on expression in CCLE and survival in neuroblastoma patients, BRD3 was identified as the top-ranked candidate. BRD3 exhibits drastic upregulation during tumor formation. Elevated BRD3 expression is the highest expressed gene in neuroblastoma cell lines upon analysis of the CCLE panel and associated with very poor prognosis. To explore the nonredundant functions of BRD3 in relation to BRD4, we performed RNA-sequencing after stable knockdown of BRD3 in neuroblastoma cell lines and compared the downstream effects on the transcriptome as well as the impact on cell viability to knockdown of BRD4 and pharmacological treatment with BET-inhibitors (JQ1, OTX015). In addition, we dissected the BRD3 protein complex by means of label-free mass spectrometry analysis to gain further insights into the BRD3 specific functions in relation to control of gene transcription and putative interaction with transcription factors such as MYCN. Current efforts are ongoing to test cooperative interaction of BRD3 versus BRD4 in dbh-MYCN driven neuroblastoma formation in zebrafish as well as BRD3 and BRD4 ChIP-sequencing in neuroblastoma cells. Conclusion: We identified BRD3 as a candidate novel driver gene in neuroblastoma and will present differential transcriptional control and protein interactions of BRD3 versus BRD4. This study can open the way towards developing BRD3 specific inhibitors for neuroblastoma and other BRD3 overexpressing cancers such as T-ALL and small cell lung carcinoma. Citation Format: Kaat Durinck, Jolien Dewyn, Anneleen Beckers, Siebe Loontiens, Suzanne Vanhauwaert, Daniel Carter, Belamy Chueng, Glenn Marshall, Katleen Depreter, Frank Westermann, Frank Speleman. BRD3 as a specific vulnerable therapeutic target in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1527. doi:10.1158/1538-7445.AM2017-1527