20 research outputs found

    Identification of novel drug targets downstream of activated ALK in neuroblastoma

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    ETV5 functionally connects ALK and CXCR4 signaling in neuroblastoma

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    Activating ALK mutations occur in 10% of neuroblastomas (NB) and represent an important druggable target for more effective treatment of high-risk patients. In order to design effective novel targeted therapeutic approaches, gaining detailed insights into downstream ALK signaling is crucial. We and others identified PI3K/AKT and RAS/MAPK as major downstream signaling axes. Also, we connected FOXO3a controlled RET expression to the PI3K/AKT axis. Here, using multiple ALK activating and inhibiting cell models, we firmly establish ETV5 as a major RAS/MAPK downstream target upregulated through mutant ALK. ETV5 is known to act as regulator of epithelial-mesenchymal transition (EMT) and controls stem cell properties and neuronal cell fate decisions. Knockdown of ETV5 reduced the clonogenic potential and growth of NB cells in vitro and in vivo. RNAseq transcriptome profiling following ETV5 knock down provided an ETV5 signature score which identifies patients with poor overall survival and was enriched in gene sets controlling EMT in keeping with observed reduced invasive properties in ETV5 depleted NB cell lines. Finally, the chemokine receptor CXCR4 emerged as a crucial ETV5 target gene thus opening unexpected novel opportunities for drugging, as CXCR4 inhibitors are available. Our data highlight ETV5 as an intrinsic component of ALK downstream and RAS/MAPK signaling in NB. The presence of RAS/MAPK and acquired ALK mutations in relapsed NB tumors highlights the significance of the ETV5 signaling pathway in NB pathogenesis. Moreover, ETV5 provides a functional link between the ALK and chemotaxis pathways involved in cancer metastasis and identifies CXCR4 as novel drug target

    HBP1 downregulation through mutant ALK represents a novel mechanism for cooperative MYCN activation in neuroblastoma

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    Introduction: MYCN signaling plays a key role in initiation and progression of neuroblastoma (NB). Mutant ALK is implicated in accelerated MYCN-driven NB formation in mouse models. Combined MYCN-amplified/mutant ALK NBs have very poor outcome. Finally, recent data showed that ALK mutations frequently occur in relapsed tumors suggesting a specific role for ALK signaling in therapy resistance. Previous studies showed that mutant ALK activates MYCN signaling through transcriptional regulation and protein stabilization. We recently identified a third major HBP1-controlled mechanism of ALK-mediated MYCN activation. Methods: Pathway analysis of HBP1 regulation was done by in vitro analysis of transcriptional response of NB cells to compounds targeting ALK, PI3K/AKT and MAPK signaling. The SHEP cell line with inducible miR-17∼92 was used to investigate HBP1 regulation. HBP1 was modulated in the NGP cell line in order to study transcriptional networks. Data mining was performed in R using available algorithms. Results: HBP1 is regulated through PI3K/AKT-FOXO3a signaling downstream of mutant ALK and a miR-17∼92 controlled negative feedback loop, as miR-17∼92 is positively regulated by MYCN and down-regulates HBP1. EGCG (epigallocatechin gallate) was selected as a tool compound as it is known to up-regulate HBP1 (Kim et al, 2006, JBC). We demonstrate synergistic effects of combined JQ1/EGCG administration in mouse xenografts with significant growth delay in this combination-treated group. Discussion: HBP1 is an important novel NB suppressor integrated into a complex regualtory network governed by ALK and MYCN signaling and offering a novel entry point for MYCN drugging in NB

    Interaction score performance.

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    <p><b>(A)</b> ROC-curve analysis of different metrics for high-throughput screening data-analysis on a set of positive and negative controls in the 3’ UTR reporter screening. Interaction scores and z-scores are calculated as described in the Methods section. B-scores are obtained by applying Tukey median polish to z-scores, in order to remove plate positional bias. Knockdowns are calculated by expressing normalized reporter activities (NRAs) relative to the average NRA of four non-targeting miRNA treated controls in the same assay plate. <b>(B)</b> Distributions of average interaction scores for positive and negative controls are clearly distinct. Application of the interaction score cutoff retrieves positive controls with 51% sensitivity, whereas negative controls are correctly called with a specificity of 99%. Precision obtained with this cutoff (i.e. the proportion of identified interactions that are true interactions) is 88%. Reprinted from Van Peer et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194017#pone.0194017.ref037" target="_blank">37</a>] under a CC-BY 4.0 license, with permission from Oxford University Press, original copyright 2016.</p