Geminin is required for Hox gene regulation to pattern the developing limb.

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5' Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5' Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb.

Uncovering the transcriptomic and epigenomic landscape of nicotinic receptor genes in non-neuronal tissues.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) play an important role in cellular physiology and human nicotine dependence, and are closely associated with many human diseases including cancer. For example, previous studies suggest that nAChRs can re-wire gene regulatory networks in lung cancer cell lines. However, the tissue specificity of nAChRs genes and their regulation remain unexplored. RESULT: In this study, we integrated data from multiple large genomic consortiums, including ENCODE, Roadmap Epigenomics, GTEx, and FANTOM, to define the transcriptomic and epigenomic landscape of all nicotinic receptor genes across many different human tissues and cell types. We found that many important nAChRs, including CHRNA3, CHRNA4, CHRNA5, and CHRNB4, exhibited strong non-neuronal tissue-specific expression patterns. CHRNA3, CHRNA5, and CHRNB4 were highly expressed in human colon and small intestine, and CHRNA4 was highly expressed in human liver. By comparing the epigenetic marks of CHRNA4 in human liver and hippocampus, we identified a novel liver-specific transcription start site (TSS) of CHRNA4. We further demonstrated that CHRNA4 was specifically transcribed in hepatocytes but not transcribed in hepatic sinusoids and stellate cells, and that transcription factors HNF4A and RXRA were likely upstream regulators of CHRNA4. Our findings suggest that CHRNA4 has distinct transcriptional regulatory mechanisms in human liver and brain, and that this tissue-specific expression pattern is evolutionarily conserved in mouse. Finally, we found that liver-specific CHRNA4 transcription was highly correlated with genes involved in the nicotine metabolism, including CYP2A6, UGT2B7, and FMO3. These genes were significantly down-regulated in liver cancer patients, whereas CHRNA4 is also significantly down-regulated in cancer-matched normal livers. CONCLUSIONS: Our results suggest important non-neuronally expressed nicotinic acetylcholine receptors in the human body. These non-neuronal expression patterns are highly tissue-specific, and are epigenetically conserved during evolution in the context of non-conserved DNA sequence.

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. Ewing's 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 Ewing's 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 Ewing's 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.

Geminin deficiency enhances survival in a murine medulloblastoma model by inducing apoptosis of preneoplastic granule neuron precursors.

Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies.

Gene regulatory networks in neural cell fate acquisition from genome-wide chromatin association of Geminin and Zic1.

Neural cell fate acquisition is mediated by transcription factors expressed in nascent neuroectoderm, including Geminin and members of the Zic transcription factor family. However, regulatory networks through which this occurs are not well defined. Here, we identified Geminin-associated chromatin locations in embryonic stem cells and Geminin- and Zic1-associated locations during neural fate acquisition at a genome-wide level. We determined how Geminin deficiency affected histone acetylation at gene promoters during this process. We integrated these data to demonstrate that Geminin associates with and promotes histone acetylation at neurodevelopmental genes, while Geminin and Zic1 bind a shared gene subset. Geminin- and Zic1-associated genes exhibit embryonic nervous system-enriched expression and encode other regulators of neural development. Both Geminin and Zic1-associated peaks are enriched for Zic1 consensus binding motifs, while Zic1-bound peaks are also enriched for Sox3 motifs, suggesting co-regulatory potential. Accordingly, we found that Geminin and Zic1 could cooperatively activate the expression of several shared targets encoding transcription factors that control neurogenesis, neural plate patterning, and neuronal differentiation. We used these data to construct gene regulatory networks underlying neural fate acquisition. Establishment of this molecular program in nascent neuroectoderm directly links early neural cell fate acquisition with regulatory control of later neurodevelopment.

Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor.

PURPOSE: The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients. EXPERIMENTAL DESIGN: We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma. RESULTS: EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo CONCLUSIONS: These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105-18. ©2016 AACR.

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.

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, O(2)-(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.

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.

A novel role for keratin 17 in coordinating oncogenic transformation and cellular adhesion in Ewing sarcoma.

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.

Promiscuous partnerships in Ewing's sarcoma.

Ewing's sarcoma is a highly aggressive bone and soft tissue tumor of children and young adults. At the molecular genetic level Ewing's sarcoma is characterized by a balanced reciprocal translocation, t(11;22)(q24;q12), which encodes an oncogenic fusion protein and transcription factor EWS/FLI. This tumor-specific chimeric fusion retains the amino terminus of EWS, a member of the TET (TLS/EWS/TAF15) family of RNA-binding proteins, and the carboxy terminus of FLI, a member of the ETS family of transcription factors. In addition to EWS/FLI, variant translocation fusions belonging to the TET/ETS family have been identified in Ewing's sarcoma. These studies solidified the importance of TET/ETS fusions in the pathogenesis of Ewing's sarcoma and have since been used as diagnostic markers for the disease. EWS fusions with non-ETS transcription factor family members have been described in sarcomas that are clearly distinct from Ewing's sarcoma. However, in recent years there have been reports of rare fusions in "Ewing's-like tumors" that harbor the amino-terminus of EWS fused to the carboxy-terminal DNA or chromatin-interacting domains contributed by non-ETS proteins. This review aims to summarize the growing list of fusion oncogenes that characterize Ewing's sarcoma and Ewing's-like tumors and highlights important questions that need to be answered to further support the existing concept that Ewing's sarcoma is strictly a "TET/ETS" fusion-driven malignancy. Understanding the molecular mechanisms of action of the various different fusion oncogenes will provide better insights into the biology underlying this rare but important solid tumor.