Savita Sankar

Microsatellites as EWS/FLI response elements in Ewing's sarcoma

The ETS gene family is frequently involved in chromosome translocations that cause human cancer, including prostate cancer, leukemia, and sarcoma. However, the mechanisms by which oncogenic ETS proteins, which are DNA-binding transcription factors, target genes necessary for tumorigenesis is not well understood. Ewings sarcoma serves as a paradigm for the entire class of ETS-associated tumors because nearly all cases harbor recurrent chromosomal translocations involving ETS genes. The most common translocation in Ewings sarcoma encodes the EWS/FLI oncogenic transcription factor. We used whole genome localization (ChIP-chip) to identify target genes that are directly bound by EWS/FLI. Analysis of the promoters of these genes demonstrated a significant over-representation of highly repetitive GGAA-containing elements (microsatellites). In a parallel approach, we found that EWS/FLI uses GGAA microsatellites to regulate the expression of some of its target genes including NR0B1, a gene required for Ewings sarcoma oncogenesis. The microsatellite in the NR0B1 promoter bound EWS/FLI in vitro and in vivo and was both necessary and sufficient to confer EWS/FLI regulation to a reporter gene. Genome wide computational studies demonstrated that GGAA microsatellites were enriched close to EWS/FLI-up-regulated genes but not down-regulated genes. Mechanistic studies demonstrated that the ability of EWS/FLI to bind DNA and modulate gene expression through these repetitive elements depended on the number of consecutive GGAA motifs. These findings illustrate an unprecedented route to specificity for ETS proteins and use of microsatellites in tumorigenesis.

Mechanism and relevance of EWS/FLI-mediated transcriptional repression in Ewing sarcoma.

Ewing sarcoma provides an important model for transcription-factor-mediated oncogenic transformation because of its reliance on the ETS-type fusion oncoprotein EWS/FLI. EWS/FLI functions as a transcriptional activator and transcriptional activation is required for its oncogenic activity. Here, we demonstrate that a previously less-well characterized transcriptional repressive function of the EWS/FLI fusion is also required for the transformed phenotype of Ewing sarcoma. Through comparison of EWS/FLI transcriptional profiling and genome-wide localization data, we define the complement of EWS/FLI direct downregulated target genes. We demonstrate that LOX is a previously undescribed EWS/FLI-repressed target that inhibits the transformed phenotype of Ewing sarcoma cells. Mechanistic studies demonstrate that the NuRD co-repressor complex interacts with EWS/FLI, and that its associated histone deacetylase and LSD1 activities contribute to the repressive function. Taken together, these data reveal a previously unknown molecular function for EWS/FLI, demonstrate a more highly coordinated oncogenic transcriptional hierarchy mediated by EWS/FLI than previously suspected, and implicate a new paradigm for therapeutic intervention aimed at controlling NuRD activity in Ewing sarcoma tumors.

GSTM4 is a microsatellite-containing EWS/FLI target involved in Ewing's sarcoma oncogenesis and therapeutic resistance

Ewings sarcoma is a malignant bone-associated tumor of children and young adults. Most cases of Ewings sarcoma express the EWS/FLI fusion protein. EWS/FLI functions as an aberrant ETS-type transcription factor and serves as the master regulator of Ewings sarcoma-transformed phenotype. We recently showed that EWS/FLI regulates one of its key targets, NR0B1, through a GGAA-microsatellite in its promoter. Whether other critical EWS/FLI targets are also regulated by GGAA-microsatellites was unknown. In this study, we combined transcriptional analysis, whole genome localization data, and RNA interference knockdown to identify glutathione S-transferase M4 (GSTM4) as a critical EWS/FLI target gene in Ewings sarcoma. We found that EWS/FLI directly binds the GSTM4 promoter, and regulates GSTM4 expression through a GGAA-microsatellite in its promoter. Reduction of GSTM4 levels caused a loss of oncogenic transformation. Furthermore, reduction of GSTM4 resulted in an increased sensitivity of Ewings sarcoma cells to chemotherapeutic agents, suggesting a role for this protein in drug resistance. Consistent with this hypothesis, patients with Ewings sarcoma whose tumors had higher levels of GSTM4 expression had worse outcomes than those with lower expression levels. These data show that GSTM4 contributes to the cancerous behavior of Ewings sarcoma and define a wider role for GGAA-microsatellites in EWS/FLI function than previously appreciated. These data also suggest a novel therapeutic resistance mechanism, in which the central oncogenic abnormality directly regulates a resistance gene.

Reversible LSD1 Inhibition Interferes with Global EWS/ETS Transcriptional Activity and Impedes Ewing Sarcoma Tumor Growth

Purpose: Ewing sarcoma is a pediatric bone tumor that absolutely relies on the transcriptional activity of the EWS/ETS family of fusion oncoproteins. While the most common fusion, EWS/FLI, utilizes lysine-specific demethylase 1 (LSD1) to repress critical tumor suppressors, small-molecule blockade of LSD1 has not yet been thoroughly explored as a therapeutic approach for Ewing sarcoma. We therefore evaluated the translational potential of potent and specific LSD1 inhibition with HCI2509 on the transcriptional program of both EWS/FLI and EWS/ERG as well as the downstream oncogenic phenotypes driven by EWS/ETS fusions in both in vitro and in vivo models of Ewing sarcoma. Experimental Design: RNA-seq was used to compare the transcriptional profiles of EWS/FLI, EWS/ERG, and treatment with HCI2509 in both EWS/FLI- and EWS/ERG-containing cell lines. We then evaluated morphologic phenotypes of treated cells with immunofluorescence. The induction of apoptosis was evaluated using caspase-3/7 activation and TUNEL staining. Colony forming assays were used to test oncogenic transformation and xenograft studies with patient-derived cell lines were used to evaluate the effects of HCI2509 on tumorigenesis. Results: HCI2509 caused a dramatic reversal of both the up- and downregulated transcriptional profiles of EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of morphologic and oncogenic phenotypes modulated by EWS/FLI. Importantly, HCI2509 displayed single-agent efficacy in multiple xenograft models. Conclusions: These data support epigenetic modulation with HCI2509 as a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins. Clin Cancer Res; 20(17); 4584–97. ©2014 AACR.

A Novel Role for Keratin 17 in Coordinating Oncogenic Transformation and Cellular Adhesion in Ewing Sarcoma

ABSTRACT Oncogenic transformation in Ewing sarcoma is caused by EWS/FLI, an aberrant transcription factor fusion oncogene. Glioma-associated oncogene homolog 1 (GLI1) is a critical target gene activated by EWS/FLI, but the mechanism by which GLI1 contributes to the transformed phenotype of Ewing sarcoma was unknown. In this work, we identify keratin 17 (KRT17) as a direct downstream target gene upregulated by GLI1. We demonstrate that KRT17 regulates cellular adhesion by activating AKT/PKB (protein kinase B) signaling. In addition, KRT17 is necessary for oncogenic transformation in Ewing sarcoma and accounts for much of the GLI1-mediated transformation function but via a mechanism independent of AKT signaling. Taken together, our data reveal previously unknown molecular functions for a cytoplasmic intermediate filament protein, KRT17, in coordinating EWS/FLI- and GLI1-mediated oncogenic transformation and cellular adhesion in Ewing sarcoma.

EWS and RE1-Silencing Transcription Factor Inhibit Neuronal Phenotype Development and Oncogenic Transformation in Ewing Sarcoma

The gene encoding EWS (EWSR1) is involved in various chromosomal translocations that cause the production of oncoproteins responsible for multiple cancers including Ewing sarcoma, myxoid liposarcoma, soft tissue clear cell sarcoma, and desmoplastic small round cell sarcoma. It is well known that EWS fuses to FLI to create EWS/FLI, which is the abnormal transcription factor that drives tumor development in Ewing sarcoma. However, the role of wild-type EWS in Ewing sarcoma pathogenesis remains unclear. In the current study, we identified EWS-regulated genes and cellular processes through RNA interference combined with RNA sequencing and functional annotation analyses. Interestingly, we found that EWS and EWS/FLI co-regulate a significant cluster of genes, indicating an interplay between the 2 proteins in regulating cellular functions. We found that among the EWS-down-regulated genes are a subset of neuronal genes that contain binding sites for the RE1-silencing transcription factor (REST or neuron-restrictive silencer factor [NRSF]), neuron-restrictive silencer element (NRSE), suggesting a cooperative interaction between REST and EWS in gene regulation. Co-immunoprecipitation analysis demonstrated that EWS interacts directly with REST. Genome-wide binding analysis showed that EWS binds chromatin at or near NRSE. Furthermore, functional studies revealed that both EWS and REST inhibit neuronal phenotype development and oncogenic transformation in Ewing sarcoma cells. Our data implicate an important role of EWS in the development of Ewing sarcoma phenotype and highlight a potential value in modulating EWS function in the treatment of Ewing sarcoma and other EWS translocation-based cancers.

Targeting glutathione S-transferase M4 in Ewing sarcoma

Ewing sarcoma is a malignant pediatric bone and soft tissue tumor. Although the 5-year survival rate of localized disease approaches 75%, the prognosis of metastatic and/or therapy-resistant disease remains dismal despite the wide use of aggressive therapeutic strategies. We previously reported that high expression of glutathione S-transferase M4 (GSTM4) in primary tumors correlates with poor patient outcomes. GSTM4 is required for oncogenic transformation and mediates resistance to chemotherapeutic drugs in Ewing sarcoma cells. Here, we performed RNA-sequencing analyses of Ewing sarcoma cells and combined our results with publicly available datasets to demonstrate that GSTM4 is a major GST specifically expressed in Ewing sarcoma. Pharmacological inhibition of GSTM4 activity using a pan GST inhibitor, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), significantly limited cellular proliferation and oncogenic transformation of Ewing sarcoma cells. Moreover, combined use of NBDHEX and etoposide synergistically increased cytotoxicity, suggesting a role for GSTM4 as an inhibitor of apoptosis. Mechanistic studies revealed that GSTM4 limits apoptosis owing to its ability to interact with Apoptosis Signal-regulating Kinase 1 (ASK1) and inhibit signaling via the c-Jun N-terminal Kinase axis. To exploit our observation that GSTM4 expression is specifically up-regulated in Ewing sarcoma, we tested the effect of a GSTM4-activated anti-cancer agent, O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate or JS-K, on tumor growth and survival. We found that JS-K robustly decreased Ewing sarcoma cell viability and xenograft tumor growth and improved overall survival of xenograft mice. Our data suggest that GSTM4 is a novel therapeutic target for the treatment of high GSTM4-expressing Ewing sarcoma. Strategies that combine standard chemotherapy with agents that inhibit GSTM4, that are activated by GSTM4, or that block GSTM4/ASK1 interactions, can potentially be more specific and/or efficacious than standard therapeutic approaches.

Abstract 3965: Modulating glutathione s-transferase M4 activity for the treatment of Ewing sarcoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ewing sarcoma is a malignant pediatric bone and soft tissue tumor. The prognosis for Ewing sarcoma remains dismal despite of intensive treatments including surgery, radiation, and chemotherapy. Moreover, these unspecific treatments often have severe side effects. We previously reported that Glutathione S-transferase M4 (GSTM4) is a potential specific treatment target for Ewing sarcoma. GSTM4 is required for oncogenic transformation and confers chemotherapeutic drug resistance in Ewing sarcoma cells; high GSTM4 level in primary tumor is correlated with poor patient outcome. Here we further evaluate the efficacy of modulating GSTM4 activity in treating Ewing sarcoma using patient-derived cells and mouse xenograft models. RNA-seq analysis for RNA levels of all GSTs in A673 Ewing sarcoma cells as well as online database searching for GSTM4 RNA level in tumor samples of various sarcomas demonstrate that GSTM4 is specifically highly expressed in Ewing sarcoma. By MTT and soft agar assays, we find that NBDHEX, a GSTM4 inhibitory compound, inhibits cellular proliferation and oncogenic transformation of Ewing sarcoma cells. Furthermore, NBDHEX has a synergistic effect in cytotoxicity with chemotherapeutic drug etoposide. Conversely, a GSTM4-activated anti-cancer agent, JS-K, significantly decreases Ewing sarcoma cell viability (p<0.05). We find that JS-K works through GSTM4 because knockdown of GSTM4 by shRNA in Ewing sarcoma cells significantly decreases their sensitivity to JS-K (p<0.05). Moreover, JS-K significantly decreases Ewing sarcoma xenograft tumor growth in immunodeficient mice (Mantel-Cox test p=0.0002). Next we examine the underlying mechanism of GSTM4 mediated drug resistance and find that GSTM4 interacts with Apoptosis Signal-regulating Kinase 1 (ASK1) and inhibits ASK1 activation of c-Jun N-terminal Kinase (JNK) mediated apoptosis upon etoposide treatment. Taken together, these data provide further evidence that GSTM4 is a novel therapeutic target for Ewing sarcoma. GSTM4 targeted inhibition by inhibitors or knockdown by RNA interference combined with standard chemotherapeutic regimens are potential treatments more specific and effective for Ewing sarcoma. GSTM4 pro-drugs are promising for treatment of patients with high GSTM4 expression tumors. Our data also suggest that agents intervening of GSTM4/ASK interaction may increase drug sensitivity of Ewing sarcoma cells and tumors and therefore be of therapeutic values. Citation Format: Rupeng Zhuo, Kenneth M. Kosak, Savita Sankar, Elizabeth T. Wiles, Yin Sun, Jianxing Zhang, Glenn D. Prestwich, Paul J. Shami, Stephen L. Lessnick, Mitchell S. Cairo, Wen Luo. Modulating glutathione s-transferase M4 activity for the treatment of 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 3965. doi:10.1158/1538-7445.AM2014-3965

Abstract 3679: Inhibition of LSD1 disrupts global EWS/ETS transcriptional function in Ewing sarcoma

Ewing sarcoma is an aggressive pediatric bone tumor characterized by an absolute reliance on the transcriptional activity of the EWS/ETS family of transcription factor fusion oncoproteins. The most common fusion is EWS/FLI, present in 85-95% of cases, though less common fusions include EWS/ERG, EWS/ETV1, EWS/ETV4 and EWS/FEV. EWS/FLI utilizes lysine-specific demethylase 1 (LSD1) as member of the NuRD complex to mediate transcriptional repression of critical tumor suppressors in Ewing sarcoma. LSD1 overexpression has been observed in clinical samples from Ewing sarcoma patients and small molecule blockade of LSD1 using tranylcypromine has been suggested as a therapeutic approach for Ewing sarcoma. We therefore evaluated the effects of LSD1 inhibition with the small molecule inhibitor, HCI-2509, in both in vitro and in vivo models of Ewing sarcoma. HCI-2509 is an LSD1 inhibitor with a Ki of ∼30 nM and multiple Ewing sarcoma cell lines show IC50s in cell viability assays from 500 nM-1 μM. Using RNA-seq, we show that HCI-2509 dramatically reverses both the up- and downregulated transcriptional profiles of both EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of oncogenic phenotypes modulated by EWS/FLI. We further developed a 9-gene panel based on the RNA-seq data to assess the transcriptional phenotype of HCI-2509 in additional cell lines and showed that it HCI-2509 disrupted both EWS/FLI-activated and -repressed genes similarly across the tested Ewing sarcoma cell lines. HCI-2509 impaired transformation of Ewing sarcoma cell lines in colony forming assays with IC50s from from 25 nM-1 μM. Notably, HCI-2509 displayed single-agent efficacy in multiple xenograft models. We investigated the PK/PD relationship using tumor histone H3K4 and H3K9 methylation. Taken together, these data suggest that epigenetic modulation through LSD1 inhibition may be a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins. Citation Format: Emily Rose Theisen, Savita Sankar, Jared Bearss, Timothy Mulvihill, Venkataswamy Sorna, Sunil Sharma, Stephen L. Lessnick. Inhibition of LSD1 disrupts global EWS/ETS transcriptional function 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 3679. doi:10.1158/1538-7445.AM2014-3679

Abstract 2219: Mechanism of trancriptional repression by EWS/FLI in Ewing's sarcoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective: Ewings sarcoma is a highly aggressive bone-associated malignancy affecting children and young adults with most cases harboring a somatic translocation that encodes the EWS/FLI oncoprotein. EWS/FLI is an aberrant transcription factor. Most studies indicate that EWS/FLI functions as a transcriptional activator. However, recent genome-wide transcriptional studies have raised the possibility that EWS/FLI may also repress target genes, although the mechanistic basis for this repression is unknown. We sought to test the hypothesis that EWS/FLI functions as a direct transcriptional repressor at some genetic loci, and that transcriptional repression is required for the oncogenic function of EWS/FLI. Furthermore, we sought to define relevant domains and corepressors that are involved in this activity. Methods: RNAi based microarray combined with genome-wide ChIP-Chip analyses were used to identify direct repressed target genes of EWS/FLI. Soft agar and xenograft tumor formation assays were performed to assess oncogenic transformation. Deletion mutants of EWS/FLI were constructed and used to map the repression domain. Co-immunoprecipitations were performed to validate the interaction of EWS/FLI with transcriptional co-repressors. Luciferase reporter assays and gel shift assays were utilized to characterize the binding site for EWS/FLI at repressed gene promoters. Results: We identified a subset of EWS/FLI-repressed target genes that are directly bound by the protein. Using these target genes, we identified domains in EWS/FLI necessary and sufficient for repression. Interestingly, these repression domains are also necessary for oncogenic transformation. We identified two HDACs that are involved in this repression, and identified a likely candidate transcriptional repression complex that binds EWS/FLI and contributes to its repressive function. Finally, we found that transcriptional repression is absolutely required for the oncogenic function of EWS/FLI, as two downregulated targets function as suppressors of Ewings sarcoma oncogenesis. Conclusion: Transcriptional deregulation by EWS/FLI is a key oncogenic event in Ewings sarcoma pathogenesis. Our data demonstrated that, in addition to transcriptional activation, EWS/FLI mediated transcriptional repression is also critical in Ewings sarcoma oncogenesis. This is the first time that a detailed molecular mechanism of EWS/FLI mediated direct repression is reported, and has important implications for therapeutic targeting of EWS/FLI in Ewings sarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2219. doi:1538-7445.AM2012-2219