Anna M. Katschnig

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.

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.

Abstract 3508: EWS-FLI1 represses Rho-actin signaling via MRTFB/YAP-1/TEAD perturbation in Ewing Sarcoma

Ewing Sarcoma (EwS) is an aggressive pediatric bone tumor driven by the aberrant fusion-oncogene EWS-FLI1, which deregulates hundreds of genes by either activation (EWS-FLI1-correlated genes) or repression (EWS-FLI1-anticorrelated genes). Several EWS-FLI1-anticorrelated genes are involved in cytoskeletal processes and typically regulated by Rho/F-actin signaling. The Rho pathway is a crucial regulator of cell movement and cellular plasticity. Given the fact that EwS cells are highly prone to metastasize we were interested in studying a potential EWS-FLI1-mediated deregulation of Rho signaling. Activation of Rho triggers G- to F-actin polymerization thereby enabling nuclear translocation of the myocardin-related transcription factors MRTFA (MKL-1) and MRTFB (MKL-2). MRTFs typically serve as transcriptional co-activators of the transcription factor SRF regulating several cytoskeletal key players. We used the A673/TR/shEF cell line, harboring a doxycycline inducible sh-EWS-FLI1 plasmid, to interrogate the role of MRTFA/B in the EwS gene regulatory network upon EWS-FLI1-high and -low conditions. Strikingly, MRTFB transcriptional activity was overall repressed by EWS-FLI1. Furthermore, knockdown of MRTFB under EWS-FLI1-low conditions antagonized the re-activation of EWS-FLI1-anticorrelated genes. ChIP-seq revealed a strong overlap of MRTFB and EWS-FLI1 chromatin binding. MRTFB binding was significantly enriched in distal (enhancer) regions of EWS-FLI1-anticorrelated genes, especially upon EWS-FLI1-low conditions. Of note, an overrepresentation of TEAD motifs, but not SRF binding motifs, was observed in these regions suggesting a potential involvement of TEAD transcription factors in the regulation of the MRTFB/EWS-FLI1 reciprocally regulated targets. In line with this finding, target genes of the mechano sensitive YAP/TAZ/TEAD pathway (CTGF, CYR61, SERPINE1) were found among the MRTFB-bound EWS-FLI1-anticorrelated genes. Genome-wide expression profiling upon combined knockdown of EWS-FLI1 and all four TEADs confirmed that TEAD regulates EWS-FLI1 target genes antagonistically. ChIP-qPCR for selected genes validated this finding demonstrating increased TEAD binding to shared MRTFB/EWS-FLI1 target genes upon decreased EWS-FLI1 occupation. Our data suggest that MRTFB/YAP-1/TEAD directly regulate EWS-FLI1-anticorrelated targets. Since, in addition, EWS-FLI1 is known to indirectly perturb F-actin polymerization, we propose a model of dual EWS-FLI1 driven MRTFB/YAP-1/TEAD perturbation by direct and indirect mechanisms downstream of Rho/F-actin signaling. Citation Format: Anna M. Katschnig, Maximilian O. Kauer, Raphaela Schwentner, Eleni M. Tomazou, Markus Linder, Cornelia N. Mutz, Javier Alonso, Dave N. Aryee, Heinrich Kovar. EWS-FLI1 represses Rho-actin signaling via MRTFB/YAP-1/TEAD perturbation in Ewing Sarcoma [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 3508. doi:10.1158/1538-7445.AM2017-3508

Abstract 1045: Targeting NAMPT in Ewing's sarcoma cells

Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in the generation of the chimeric transcription factor EWS-FLI1. NAD is a key metabolite of energy metabolism being involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability serving as a donor of ADP-ribose. This study describes targeting NAMPT (nicotinamide phosphoribosyltransferase), the rate-limiting enzyme in salvage generation of NAD, by FK866 in ES cells. Using FK866 has been proposed as a treatment option for various inflammatory diseases as well as cancer, rendering ES cells with high NAMPT expression especially susceptible to FK866-induced cytotoxicity. Here we report that NAMPT inhibition in ES cells leads to exhaustive NAD depletion, followed by a delayed reduction of ATP levels and concomitantly to apoptosis-mediated cell death. These effects can be reversed by nicotinic acid, a substrate for the NAD salvage generation. However, the use of a doxycycline-inducible shRNA against EWS-FLI1 revealed that the cytotoxic activity of NAMPT inhibition is significantly lowered in the absence of EWS-FLI1. EWS-FLI1-low ES cells have higher viability and lower rates of apoptosis throughout inhibitor treatment compared to cells with high EWS-FLI1 expression. Additionally, changes in mitochondrial respiration and glycolytic rate can be observed when comparing untreated versus EWS-FLI1 knockdown ES cells after NAMPT inhibition. Interestingly, loss of EWS-FLI1 leads to elevated NAD levels and results in alteration of RNA expression of some enzymes involved in the NAD synthesis pathway. These results might explain the high susceptibility of Ewing Sarcoma cells to FK866 treatment. Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of ES cells and suggests NAMPT inhibition as a potential new treatment approach for Ewing Sarcoma in combination with standard therapies. Supported by the Austrian Science fund, grant I1225-B19; and the Research Manitoba and CancerCare Manitoba Foundation. Citation Format: Cornelia N. Mutz, Raphaela Schwentner, Eric Bouchard, Edgard M. Mejia, Anna M. Katschnig, Maximilian O. Kauer, Dave N.T. Aryee, Antje Garten, Versha Banerji, Heinrich Kovar. Targeting NAMPT in Ewing9s sarcoma cells. [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 1045.

Abstract 2105: An EWS-FLI1/MRTF gene regulatory network in Ewing sarcoma

The pediatric bone cancer Ewing sarcoma (ES) is characterized by the expression of the chimeric transcription factor EWS-FLI1, derived from a chromosomal translocation t(11;22)(q24;q12). The early onset of metastasis in ES leads to poor overall survival and limited treatment options. Remodeling of the actin cytoskeleton is a prerequisite of a tumor cell in order to become metastatic. Previous studies suggest that EWS-FLI1 alters cell morphology and deregulates various cytoskeletal genes. The major point of convergence of extracellular signals influencing cytoskeletal regulation is the Rho pathway. Activation of Rho GTPases promotes polymerization of monomeric G-actin into stress fibers (F-actin) thereby releasing the myocardin-related transcription factor family (MRTF-A, MRTF-B) of transcriptional coactivators. MRTFs translocate to the nucleus and interact with the transcription factor serum response factor (SRF) bound on CArG promoter elements of genes. However, the ternary complex factor (TCF) family, a class of transcriptional coactivators activated downstream of Ras signaling, competes with MRTFs for SRF binding in presence of ets binding sites adjacent to the CArG consensus sequence. The ets-transcription factor EWS-FLI1 has been shown to substitute for TCFs and interact with SRF. We hypothesized that EWS-FLI1 competes with MRTFs for SRF binding and uncouples target gene expression from Rho signaling. In combinatorial knockdown experiments of MRTFA/B and EWS-FLI1 we demonstrate that global changes in gene expression elicited by EWS-FLI1 silencing largely depend on the presence of MRTFs, suggesting that the fusion oncogene prohibits MRTF activity. Using gene expression analysis we identified six different patterns of MRTF/EWS-FLI1 interaction, with predominance of EWS-FLI1 inversely regulating MRTF target genes. Furthermore, we found MRTF transcriptional effects are already present in the absence of serum in A673 ES cells. MRTFB, however, is cytoplasmic under serum starved conditions and translocates to the nucleus only upon serum stimulation unless latrunculin B (LatB), an actin polymerization inhibitor, is added. In contrast, MRTFA is constitutively nuclear even under serum starved conditions or upon LatB treatment indicating that MRTFA might be uncoupled from actin regulation. These findings reveal a previously unknown role of MRTFs and the “CArGome” in EWS-FLI1 driven transcriptional dysregulation. Supported by the Liddy Shriver Sarcoma Initiative. Citation Format: Anna M. Katschnig, Maximilian Kauer, Raphaela Schwentner, Dave N.T Aryee, Elizabeth Lawlor, Heinrich Kovar. An EWS-FLI1/MRTF gene regulatory network in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2105. doi:10.1158/1538-7445.AM2015-2105

Abstract 1407: The influence of EWS-FLI1 on the Rho/Actin/MRTF circuit in Ewing sarcoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ewing sarcoma (ES) is the second most common bone tumor in children and adolescents. The disease is characterized by the expression of the aberrant transcription factor EWS-FLI1, which is a fusion gene product originating from the chromosomal translocation t(11;22)(q24;q12). Despite good treatment outcomes for patients with localized ES, about 25% of patients already harbor metastatic disease upon presentation and are prevalently faced with poor overall survival. The metastatic cascade is a complex process. Remodeling of the actin cytoskeleton is the first step for a cell in order to acquire a migratory phenotype. The major regulator of the actin cytoskeleton is the Rho family of GTPases, which upon G-protein coupled receptor (GPCR) signaling gets activated and promotes the polymerization of monomeric G-actin into stress fibers. Concurrently G-actin releases the myocardin-related transcription factor family (MRTF) of transcriptional coactivators. MRTFs translocate into the nucleus where they, together with the transcription factor serum response factor (SRF), bind to serum response elements (SRE) in the promoter of genes involved in cell adhesion, migration and motility. However, the ternary complex factor (TCF) family, a class of transcriptional coactivators activated through Ras signaling, can compete with MRTFs for SRF complexing and binds to ets-motifs on SRE. The ets-transcription factor EWS-FLI1 has been shown to substitute for TCFs, potentially interfering with SRF target gene expression. We therefore hypothesize that EWS-FLI1 interferes with GPCR downstream signaling by competition with MRTFs. By gene expression profiling, we show that several putative MRTF target genes are repressed in the presence of EWS-FLI1 in ES cell lines and are strongly induced upon EWS-FLI1 knockdown. Using the MRTFA target gene transgelin (TAGLN) as an example, we show that knockdown of MRTFA can only reduce its serum inducibility in the absence but not in the presence of EWS-FLI1 supporting our hypothesis of EWS-FLI1 perturbing GPCR signaling in ES. With this study we want to elucidate the role of EWS-FLI1 in the regulation of early steps of the metastatic process in ES. Supported by the Liddy Shriver Sarcoma Initiative. Citation Format: Anna M. Katschnig, Raphaela Schwentner, Stephan Niedan, Maximilian O. Kauer, Elizabeth R. Lawlor, Dave N.T. Aryee, Heinrich Kovar. The influence of EWS-FLI1 on the Rho/Actin/MRTF circuit in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1407. doi:10.1158/1538-7445.AM2014-1407