Dave N.T. Aryee

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL‐2H3) have previously been shown to contain a single type of voltage‐activated channel, namely an inwardly rectifying K+ channel, under normal recording conditions. Thus, RBL‐2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K+ channels. Injection of mRNA isolated from RBL‐2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K+ equilibrium potential (EK), (2)decreased in slope conductance near EK, (3) was dependent on [K+]o and (4) was blocked by external Ba2+ and Cs+. These properties were similar to those of the inwardly rectifying K+ current recorded from RBL‐2H3 cells using whole‐cell voltage clamp. Injection of size‐fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K+ channels.

Factors Affecting EWS-FLI1 Activity in Ewing's Sarcoma

Ewings sarcoma family tumors (ESFT) are characterized by specific chromosomal translocations, which give rise to EWS-ETS chimeric proteins. These aberrant transcription factors are the main pathogenic drivers of ESFT. Elucidation of the factors influencing EWS-ETS expression and/or activity will guide the development of novel therapeutic agents against this fatal disease.

Single- chain antibodies to the EWS NH2 terminus structurally discriminate between intact and chimeric EWS in ewing's sarcoma and interfere with the transcriptional activity of EWS in vivo

The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewings sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH(2) terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4- and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies.

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.

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.

Abstract 2028: Transcriptional modules involving EWS-FLI1 and SRF in Ewing sarcoma

Ewing Sarcoma (ES) is an aggressive pediatric bone tumor with a high tendency to metastasize. The primary genetic aberration responsible for ES pathogenesis is EWS-FLI1, a chimeric gene, derived from the chromosomal translocation t(11;22)(q24;q12). EWS-FLI1 encodes an aberrant ETS transcription factor. However, due to its lack of enzymatic activity and intrinsically disordered structure it has yet not been successfully targeted. EWS-FLI1 is prone to rapidly engage in protein-protein complexes, enhancing its oncogenic activity. It has been reported to be involved in interactions with regulators of the basal transcriptional machinery, RNA processing and DNA repair. Furthermore, it has been shown that, similar to the ternary complex factors (TCF) SAP1a and ELK1, EWS-FLI1 is able to form a complex with the global transcriptional regulator Serum Response Factor (SRF) on ETS containing promoter elements. SRF binds to CArG motifs in serum response elements (SRE) and requires a transcriptional co-activator, which is commonly provided upstream via the Rho- or the Ras-axis. Upon serum induced Rho activation, polymerization of globular (G-) actin into filamentous (F-) actin takes place, thereby releasing the G-actin bound myocardin-related transcription factors A and B (MRTFA/B) to translocate to the nucleus and concertedly with SRF regulate transcription of target genes. Binding of MRTFs to SRF is mutually exclusive and competed by TCFs, which are activated downstream of Ras, and require presence of an ETS motif adjacent to CArG. In this study we aim to investigate a potential competition of EWS-FLI1 with MRTFs, which might uncouple SRF dependent transcription from upstream G-protein linked regulation and hence interfere with processes such as proliferation, migration and differentiation. To address this question, we performed gene expression profiling in A673 and SKNMC ES cell lines where we combined EWS-FLI1 knockdown with MRTFA/B knockdown under serum starved and serum induced (SI) conditions. We found that transcriptional activity of MRTFA/B is largely repressed by EWS-FLI1 and that upon combined knockdown conditions, EWS-FLI1 transcriptional effects are rescued by MRTFA/B. To further elucidate the underlying mechanisms of EWS-FLI1 and MRTFA/B gene regulation, we performed chromatin immunoprecipitation combined with massive parallel sequencing (ChIP-seq) of MRTFA, MRTFB, SRF and EWS-FLI1 in A673 ES cells. Preliminary results corroborate EWS-FLI1 and MRTF sharing a large number of target genes and indicate that MRTFA binds to DNA motifs, which are associated with activated and repressive EWS-FLI1 signatures. Ongoing experiments aim at a proteomic understanding of the EWS-FLI1 containing SRF complex as a basis for potential therapeutic intervention. This study was supported by the Liddy Shriver Sarcoma Initiative and the 7th framework program of the European Commission (FP7-259348, “ASSET”). Citation Format: Anna M. Katschnig, Maximilian O. Kauer, Dave N t Aryee, Raphaela Schwentner, Elizabeth Lawlor, Heinrich Kovar. Transcriptional modules involving EWS-FLI1 and SRF in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2028.

Abstract 444: Hypoxia modulates EWS-FLI1 transcriptional signature and enhances invasiveness and anchorage-independent growth of Ewing's sarcoma cells in-vitro

Hypoxia is an important factor in the tumor cell microenvironment and approximately 1-1.5% of the genome is transcriptionally responsive to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is the major mediator of transcriptional activation under hypoxia. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. By immunohistochemistry, HIF-1α expression was readily detectable in 18/28 primary Ewing′s sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, hence we studied the effect of hypoxia on ESFT cell lines in-vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is transiently up-regulated by hypoxia in a HIF-1α-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis related genes, hypoxia stimulated the invasiveness and soft-agar colony formation of ESFT cells in-vitro. Our data represents the first transcriptome analysis of hypoxic ESFT cells and identifies hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity that may impact the malignant properties of ESFT. This study was funded by: “European Embryonal Tumor Pipeline” 6 th framework program of the European Commission (STREP “E.E.T. Pipeline” contract LSHC-CT-2006-037260) and grant 12675 from the Austrian National Bank Jubilaeumsfonds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 444.

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.

Abstract A02: Targeting EWS-FLI1 dose-dependent epithelial-to-mesenchymal transition

Reversible transitions from a proliferative to a nonproliferative but highly migratory and invasive phenotype, widely known as epithelial-to-mesenchymal transition (EMT), is a prerequisite for wound healing and organ development but also a hallmark of cancer. Recently, phenotypic changes resembling EMT were described to occur in Ewing sarcoma upon modulation of expression of the driver oncogene EWS-FLI1. Evidence was provided for the presence of highly migratory EWS-FLI1-low cells in Ewing sarcoma cell lines as well as in about 20% of primary tumors at low frequency (Franzetti et al., 2017). Importantly, when injected into the tail vein of immunosuppressed mice, EWS-FLI1-low cells were more likely to result in lung metastases than EWS-FLI1-high cells upon re-expression of the oncogene. In our study, we identified a mechanism for the cytoskeletal reprograming underlying EWS-FLI1 dose-dependent phenotypic transitions in Ewing sarcoma. We provide evidence for EWS-FLI1 blocking access of the transcriptional coactivators MRTFB and TAZ to the YAP/TEAD transcription factor complex on enhancers of EWS-FLI1 anti-correlated genes. Upon modulation of EWS-FLI1 expression, TEADs get activated downstream of Rho signaling through recruitment of MRTFB/YAP/TAZ, leading to the expression of key cytoskeletal components and regulators involved in the reorganization of the actin cytoskeleton. Based on these findings, we hypothesized that small-molecule inhibitors of the YAP/TAZ/TEAD pathway should interfere with the migratory phenotype of EWS-FLI1-low cells. In fact, the YAP/TAZ inhibitor verteporfin (Visudyne®), which is clinically used as a photosensitive drug in the treatment of age-related macular degeneration, was able to block Ewing sarcoma cell migration at low nanomolar concentrations without affecting tumor cell proliferation. These results suggest that YAP/TAZ inhibitors may be considered as a promising add-on to conventional combination treatments to prohibit tumor cell dissemination and metastasis. Citation Format: Anna M. Katschnig, Maximilian O. Kauer, Raphaela Schwentner, Eleni M. Tomazou, Dave N.T. Aryee, Heinrich Kovar. Targeting EWS-FLI1 dose-dependent epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A02.

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.