Identifying the druggable interactome of EWS-FLI1 reveals MCL-1 dependent differential sensitivities of Ewing sarcoma cells to apoptosis inducers

Ewing sarcoma (EwS) is an aggressive pediatric bone cancer in need of more effective therapies than currently available. Most research into novel targeted therapeutic approaches is focused on the fusion oncogene EWSR1-FLI1, which is the genetic hallmark of this disease. In this study, a broad range of 3,325 experimental compounds, among them FDA approved drugs and natural products, were screened for their effect on EwS cell viability depending on EWS-FLI1 expression. In a network-based approach we integrated the results from drug perturbation screens and RNA sequencing, comparing EWS-FLI1-high (normal expression) with EWS-FLI1-low (knockdown) conditions, revealing novel interactions between compounds and EWS-FLI1 associated biological processes. The top candidate list of druggable EWS-FLI1 targets included genes involved in translation, histone modification, microtubule structure, topoisomerase activity as well as apoptosis regulation. We confirmed our in silico results using viability and apoptosis assays, underlining the applicability of our integrative and systemic approach. We identified differential sensitivities of Ewing sarcoma cells to BCL-2 family inhibitors dependent on the EWS-FLI1 regulome including altered MCL-1 expression and subcellular localization. This study facilitates the selection of effective targeted approaches for future combinatorial therapies of patients suffering from Ewing sarcoma.(VLID)471264

Investigating the NAD metabolome in ewing sarcoma

Ewing-Sarkom (ES) ist der zweithäufigste Knochentumor nach Osteosarkom und tritt bevorzugt im Kindes- und Jugendalter auf. In 85% der Fälle lässt sich die Ursache der Erkrankung durch eine chromosomale Translokation t(11;22)(q24;12) erklären, welche zur Bildung eines chimären Transkriptionsfaktors führt. Diese Doktorarbeit beschäftigt sich mit der Analyse von zwei miteinander verwobenen Stoffwechselwegen in ES, die unter dem Namen “NAD-Metabolom” zusammen gefasst werden. Nikotinamidadenindinukleotid (NAD) ist ein äußerst wichtiger Metabolit, der für den Energiehaushalt der Zelle unerlässlich ist. NAD kann entweder ausgehend von der essentiellen Aminosäure Tryptophan (TRP) de novo oder durch effizientere Regeneration von Vitamin B3-Derivaten synthetisiert werden. Nikotinamidphosphoribosyltransferase (NAMPT) ist das geschwindigkeitsbestimmende Enzym in der NAD-Synthese von Säugetieren und ist in ES-Zellen stark exprimiert. Im Allgemeinenen zeichnen sich Krebszellen durch einen veränderten Metabolismus aus und somit stellt das Eingreifen in den Tumormetabolismus eine vielversprechende neue Krebstherapie dar. Wegen des erhöhten Energiebedarfs von Krebszellen sind diese besonders empfindlich für verminderte Verfügbarkeit von NAD. In unserer Studie haben wir den spezifischen NAMPT-Inhibitor FK866 verwendet und konnten damit zeigen, dass durch NAD-Verbrauch in den Zellen ein metabolischer Kollaps induziert wird. Dieser geht mit der Blockade von Glykolyse und Fehlfunktion von Mitochondrien einher, führt zu einer Verringerung von Adenosintriphosphat und zum Tod der Zellen. Gerade Zellen, die EWS-FLI1 exprimieren, sind besonders empfindlich auf NAMPT-induzierte NAD-Reduktion. Dies haben wir in A673sh-Zellen untersucht, in denen EWS-FLI1-Expression durch RNA-Interferenz (RNAi) verringert werden kann. ^Der Kynurenin-Signalweg stellt den ersten Teil der NAD de novo Synthese dar, ausgehend von der Aminosäure TRP, welche von Tryptophan 2,3-Dioxygenase (TDO2) oxidiert wird. Einige Metabolite aus den ersten Schritten des TRP-Abbaus sind in den ES-Zellen A673sh unter geringer EWS-FLI1-Expression stark hochreguliert. Dies betrifft im Besonderen Kynurenin (KYN) und Kynureninsäure (KYNA), welche stark akkumulieren. Wir konnten zeigen, dass KYN- und KYNA-Anhäufung zur Aktivierung des Arylhydrokarbonrezeptors (AHR) führten. AHR bindet and Dioxin-response Elemente (DRE) in der Promoterregion von Ziel-Genen und erhöht die Expression von den Genen IL8, IL6, IL1B, CYP1A1, CYP1B1, TUFT1 und FAM65B wenn wenig EWS-FLI1-Expression vorhanden ist. Normalerweise supprimiert EWS-FLI1 den autokrinen AHR-Signaltransduktionsweg in A673sh-Zellen. Unsere Ergebnisse deuten auf einen regulatorischen Effekt von AHR-Aktivierung in A673sh-Zellen hin, im Besondern bei geringer EWS-FLI1-Expression.Ewing Sarcoma (ES) is the second most common bone cancer occurring in children and adolescents with a peak incidence at the age of 15. In 85% of tumors, the driving force of the malignancy is the chimeric transcription factor EWS-FLI1, resulting from the specific chromosomal translocation t(11;22)(q24;q12). In this dissertation, I report the investigation of two interconnected pathways, both contributing to the “NAD metabolome” of ES. Nicotinamide adenine dinucleotide (NAD) is a key metabolite essential for sustaining cellular energy metabolism and necessary for glycolytic and mitochondrial function, DNA repair, genomic stability, chromatin remodeling, and signal transduction. NAD can either be synthesized de novo from the essential amino acid tryptophan (TRP), or much more efficiently via salvage pathways starting from derivatives of vitamin B3. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of mammalian NAD salvage synthesis and is highly expressed in ES cells. Targeting tumor cell metabolism has become an attractive anti-cancer approach and given the increased metabolic needs of cancer cells, they are supposed to be hit especially hard by NAD depletion. We used the small molecule compound FK866 for inhibition of NAMPT and could show that upon excessive NAD exhaustion cells enter acute metabolic stress such as glycolytic impairment, mitochondrial dysfunction, adenosine triphosphate (ATP) depletion and finally cell death. Especially cells expressing EWS-FLI1 are exquisitely sensitive to NAMPT inhibition in contrast to cells expressing lower levels of EWS-FLI1 as investigated in RNAi-inducible A673sh cells. The kynurenine pathway is part of NAD de novo synthesis from TRP which is oxidized by tryptophan 2,3-dioxygenase (TDO2). By analyzing several metabolites of the initial steps of TRP degradation, we observed that A673sh cells with low EWS-FLI1 expression strongly enhanced TRP breakdown in favor of kynurenine (KYN) and kynurenic acid (KYNA) production. Strikingly, the downstream effects of KYN and KYNA accumulation led to activation of the aryl hydrocarbon receptor (AHR), a ligand-activated cytoplasmic transcription factor. AHR binds to dioxin-response elements (DRE) in the promoter region of target genes and up-regulates IL8, IL6, IL1B, CYP1A1, CYP1B1, TUFT1, and FAM65B under conditions with low EWS-FLI1 expression. We therefore suggest an involvement of AHR for an alternative survival strategy of A673sh cells with depleted EWS-FLI1 expression. Our data reveal that EWS-FLI1 usually suppresses autocrine AHR signaling by impairing TDO2-mediated TRP breakdown.submitted by Corneloa Noëlle Mutz, MSc.Zusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)171462

The role of miR-17-92 in the miRegulatory landscape of Ewing sarcoma

MicroRNAs serve to fine-tune gene expression and play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a tissue still poses a significant problem since the presence of a seed sequence in the 3'UTR of an mRNA and its expression modulation upon ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing sarcoma. MiR-17-92, one of the most potent oncogenic miRNAs, was recently reported to be among the top EWS-FLI1 activated miRNAs. Using a combination of AGO2 pull-down experiments by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) and of RNAseq upon miRNA depletion by ectopic sponge expression, we aimed to identify the targetome of miR-17-92 in Ewing sarcoma. Intersecting both datasets we found an enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3'UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Strikingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signaling. Testing for SMAD phosphorylation, we identify quiet but activatable TGFB signaling and cell autonomous activity of the BMP pathway resulting in the activation of the stemness regulatory transcriptional repressors ID1 and ID3. Taken together, our findings shed light on the complex miRegulatory landscape of Ewing Sarcoma pointing miR-17-92 as a key node connected to TGFB/BMP pathway.This study was supported in part by the Austrian Science Fund (FWF), [grants 24708-B21 and I1225-B19], and by the 7th framework program of the European Commission, [grant FP7-259348] (‘ASSET’).S

EWS-FLI1 confers exquisite sensitivity to NAMPT inhibition in Ewing sarcoma cells

Ewing sarcoma (EwS) is the second most common bone cancer in children and adolescents with a high metastatic potential. EwS development is driven by a specific chromosomal translocation resulting in the generation of a chimeric EWS-ETS transcription factor, most frequently EWS-FLI1.Nicotinamide adenine dinucleotide (NAD) is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. This study describes targeting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD synthesis, by FK866 in EwS cells. Here we report that blocking NAMPT leads to exhaustive NAD depletion in EwS cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shRNA revealed that EWS-FLI1 depletion significantly reduces the sensitivity of EwS cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 EwS cell lines of different genotypes with 5 Non-EwS cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive EwS cells, with IC50 values mostly below 1nM.Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of EwS cells and suggest NAMPT inhibition as a potential new treatment approach for Ewing sarcoma.Austrian Science Fund (FWF), grant I1225-B19; European Union's FP7 project ASSET (grant agreement No. 259348); Research Manitoba and CancerCare Manitoba Foundation. Heart and Stroke Foundation of Canada (G-14-0005708). E.M.M. is the recipient of a Research Manitoba Graduate Studentship. G.M.H. is the Canada Research Chair in Molecular Cardiolipin Metabolism. J.A. is supported by Asociación Pablo Ugarte and Miguelañez S.A, ASION-La Hucha de Tomás, Fundación La Sonrisa de Alex and Instituto de Salud Carlos III (PI12/00816 and Spanish Cancer Network RTICC RD12/0036/0027).S

FEBS Letters / EWS-FLI1 impairs aryl hydrocarbon receptor activation by blocking tryptophan breakdown via the kynurenine pathway

Ewing sarcoma (ES) is an aggressive pediatric tumor driven by the fusion protein EWS-FLI1. We report that EWS-FLI1 suppresses TDO2-mediated tryptophan (TRP) breakdown in ES cells. Gene expression and metabolite analyses reveal an EWS-FLI1-dependent regulation of TRP metabolism. TRP consumption increased in the absence of EWS-FLI1, resulting in kynurenine and kynurenic acid accumulation, both aryl hydrocarbon receptor (AHR) ligands. Activated AHR binds to the promoter region of target genes. We demonstrate that EWS-FLI1 knockdown results in AHR nuclear translocation and activation. Our data suggest that EWS-FLI1 suppresses autocrine AHR signaling by inhibiting TDO2-catalyzed TRP breakdown.I 1225-B19(VLID)296348

Combinatorial Drug Screening Identifies Ewing Sarcoma–specific Sensitivities

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma -specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1. We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1 dependent manner

Challenges and technological approaches for tackling emerging contaminants in drinking and wastewater

In recent decades, emerging contaminants (ECs) have surfaced as one of the key environmental problems threatening ecosystems and public health. Most emerging contaminants are present in low concentrations, and therefore often remain undetected and are also referred to as ‘micropollutants’. Despite this, many ECs raise considerable concerns regarding their impacts on human and environmental health. DEMEAU (Demonstration of promising technologies to address emerging contaminants in water and wastewater), a European Seventh Framework Programme (EU-FP7, 2013-2015) project, aimed to tackle ECs in drinking and wastewater by advancing the uptake of knowledge, prototypes, practices and removal technologies. The project followed a solutions-oriented approach using applied research and demonstration sites, and explored four promising technologies for EC removal and/or degradation: Managed Aquifer Recharge (MAR), Hybrid Ceramic Membrane Filtration (HCMF), Automatic Neural Net Control Systems (ANCS) and Advanced Oxidation Techniques (AOT). Furthermore, Bioassays (BA) were investigated as an effect-based monitoring tool. This article shares new findings for each approach and their potential for widespread integration in the drinking- and wastewater sector. Research results from DEMEAU demonstration sites show that opportunities for synergies among these developments offer the most promising and effective methods for tackling ECs in the water sector